Tubulin Research Articles

Mutation of the SUMOylation site of Aurora-B disrupts spindle formation and chromosome alignment in oocytes

Aurora-B is a kinase that regulates spindle assembly and kinetochore-microtubule (KT-MT) attachment during mitosis and meiosis. SUMOylation is involved in the oocyte meiosis regulation through promoting spindle assembly and chromosome segregation, but its substrates to support this function is still unknown. It is reported that Aurora-B is SUMOylated in somatic cells, and SUMOylated Aurora-B contributes the process of mitosis. However, whether Aurora-B is SUMOylated in oocytes and how SUMOylation of Aurora-B impacts its function in oocyte meiosis remain poorly understood. In this study, we report that Aurora-B is modified by SUMOylation in mouse oocytes. The results show that Aurora-B colocalized and interacted with SUMO-2/3 in mouse oocytes, confirming that Aurora-B is modified by SUMO-2/3 in this system. Compared with that in young mice, the protein expression of SUMO-2/3 decreased in the oocytes of aged mice, indicating that SUMOylation might be related to mouse aging. Overexpression of Aurora-B SUMOylation site mutants, Aurora-BK207R and Aurora-BK292R, inhibited Aurora-B recruitment and first polar body extrusion, disrupting localization of gamma tubulin, spindle formation and chromosome alignment in oocytes. The results show that it was related to decreased recruitment of p-HDAC6 which induces the high stability of whole spindle microtubules including the microtubules of both correct and wrong KT-MT attachments though increased acetylation of microtubules. Therefore, our results corroborate the notion that Aurora-B activity is regulated by SUMO-2/3 in oocytes, and that SUMOylated Aurora B plays an important role in spindle formation and chromosome alignment.

EPIGENETIC MODULATION OF STEMNESS AND DIFFERENTIATION BY VALPROIC ACID IN MEDULLOBLASTOMA

Abstract AIMS Changes in epigenetic processes, including histone acetylation, are proposed as key events affecting tumor cell function and the initiation and progression of pediatric brain cancers. Valproic acid (VPA) is an antiepileptic drug that acts partially by inhibiting histone deacetylases (HDACs) and could be repurposed as an epigenetic anticancer therapy. Here, we report VPA-induced alterations in features related to stemness and differentiation in medulloblastoma (MB) cells. METHOD Human MB cells were treated with different concentrations of VPA and cell viability was measured with a trypan exclusion assay. Cell cycle was evaluated with flow cytometry. Expansion of MB cancer stem cells (CSCs) was evaluated by a neurosphere formation assay. Reverse transcriptase polymerase chain reaction (RT-qPCR) was used to analyze mRNA expression and Western Blot to analyze protein levels. H3K9 histone acetylation (H3K9ac) was measured with immunofluorescence, and chromatin Immunoprecipitation (ChIP) was used to evaluate the occupancy of gene promoter regions by H3K9ac. RESULTS VPA reduced MB cell viability and expansion of MB CSCs, induced morphological changes consistent with neuronal differentiation, and increased expression of differentiation markers tubulin beta 3 class III (TUBB3) and enolase 2 (ENO2). Expression of stemness markers SOX2 and Nestin was differentially affected by VPA in cells representative of SHH or Group 3/4 MB molecular subgroups. VPA increased H3K9ac and its occupancy of promoter regions of TP53 and CDKN1A genes. CONCLUSION Our results indicate that VPA may exert antitumor effects in MB by influencing histone acetylation, which may result in modulation of stemness and stimulation of neuronal differentiation.

A Taxonomic and Phylogenetic Study of Anamorphic Strains of Daldinia (Hypoxylaceae, Xylariales) in Southern China.

In an extensive fungal investigation conducted in southern China, a large number of fungal strains were isolated by collecting and treating diseased and decayed leaves. Using internal transcribed spacer regions (ITSs) sequence data for a BLAST search to screen for suspected strains of Daldinia, followed by phylogenetic analysis using internal transcribed spacer regions, partial sequences of the large subunit of the rDNA (LSU), RNA polymerase II (rpb2), and beta tubulin (tub2) sequence data, combined with morphological characteristics of anamorphic species, ninety-four strains of Daldinia were identified. Furthermore, their geographical distribution and host specificity of the genus were thoroughly analyzed and summarized. Additionally, seven new anamorphic species of the genus Daldinia were also detected, Daldinia ehretiae sp. nov., D. jianfengensis sp. nov., D. ledongensis sp. nov., D. menghaiensis sp. nov., D. rhododendri sp. nov., D. spatholobi sp. nov., and D. thunbergiae sp. nov.

Optimization of Fermentation Conditions of Cordyceps militaris and In Silico Analysis of Antifungal Property of Cordycepin Against Plant Pathogens.

Cordyceps militaris, a medicinal fungus, has gained considerable attention owing to its potential health benefits, notably the production of bioactive compounds such as cordycepin. Cordycepin possesses significant antifungal, antibacterial, and antiviral properties. The present study focused on optimizing the fermentation conditions for C.militaris to boost the production of mycelia and cordycepin, alongside investigating its antifungal properties using in silico and in vitro approaches. The optimal conditions, yielding the highest cordycepin and mycelial biomass, were a temperature of 20°C and a pH range of 4-6, with glucose and sucrose as carbon sources and yeast extract and casein hydrolysate as nitrogen sources. Under these conditions, cordycepin production peaked at low pH (600-1000 mg/L) and with carbon and maltose (400-500 mg/L). The low temperature favored cordycepin production (400 mg/L), whereas casein hydrolysate as a nitrogen source boosted cordycepin yield (600 mg/L). The docking analysis indicated that cordycepin had the highest binding affinity for the tubulin beta chain 2 (-10.4 kcal/mol) compared to the fungicide tebuconazole (-7.9 kcal/mol for both targets). The in silico results were corroborated by in vitro studies, where the mycelial extract of C.militaris inhibited approximately 75% of fungal growth at a concentration of 6000 ppm. These findings suggest that optimizing fermentation conditions significantly enhances cordycepin production, and cordycepin shows antifungal solid activity, making it a promising agent for biocontrol in agriculture.

Sabizabulin Ameliorates Kidney Fibrosis by Inhibition of Myofibroblast-Specific Beta Tubulin 3

Sabizabulin Ameliorates Kidney Fibrosis by Inhibition of Myofibroblast-Specific Beta Tubulin 3

A patient-derived cell model for malignant transformation in IDH-mutant glioma

Malignant transformation (MT) is commonly seen in IDH-mutant gliomas. There has been a growing research interest in revealing its underlying mechanisms and intervening prior to MT at the early stages of the transforming process. Here we established a unique pair of matched 3D cell models: 403L, derived from a low-grade glioma (LGG), and 403H, derived from a high-grade glioma (HGG), by utilizing IDH-mutant astrocytoma samples from the same patient when the tumor was diagnosed as WHO grade 2 (tumor mutational burden (TMB) of 3.96/Mb) and later as grade 4 (TMB of 70.07/Mb), respectively. Both cell models were authenticated to a patient’s sample retaining endogenous expression of IDH1 R132H. DNA methylation profiles of the parental tumors referred to LGG and HGG IDH-mutant glioma clusters. The immunopositivity of SOX2, NESTIN, GFAP, OLIG2, and beta 3-Tubulin suggested the multilineage potential of both models. 403H was more prompt to cell invasion and developed infiltrative HGG in vivo. The differentially expressed genes (DEGs) from the RNA sequencing analysis revealed the tumor invasion and aggressiveness related genes exclusively upregulated in the 403H model. Pathway analysis showcased an enrichment of genes associated with epithelial-mesenchymal transition (EMT) and Notch signaling pathways in 403H and 403L, respectively. Mass spectrometry-based targeted metabolomics and hyperpolarized (HP) 1-13C pyruvate in-cell NMR analyses demonstrated significant alterations in the TCA cycle and fatty acid metabolism. Citrate, glutamine, and 2-HG levels were significantly higher in 403H. To our knowledge, this is the first report describing the development of a matched pair of 3D patient-derived cell models representative of MT and temozolomide (TMZ)-induced hypermutator phenotype (HMP) in IDH-mutant glioma, providing insights into genetic and metabolic changes during MT/HMP. This novel in vitro model allows further investigation of the mechanisms of MT at the cellular level.Graphic

TUBB4A Inhibits Glioma Development by Regulating ROS-PINK1/Parkin-Mitophagy Pathway.

Glioma is a refractory malignant tumor with a powerful capacity for invasiveness and a poor prognosis. This study aims to investigate the role and mechanism of tubulin beta class IVA (TUBB4A) in glioma progression. The differential expression of TUBB4A in humans was obtained from databases and analyzed. Glioma cells U251-MG and U87-MG were intervened by pcDNA3.1(+) and TUBB4A overexpression plasmid. MTT, CCK8, LDH, wound healing, transwell, and western blotting were used to explore whether TUBB4A participates in the development of glioma. Reactive oxygen species (ROS) were detected by the DCFH-DA probe. Mitochondrial membrane potential (MMP) was examined by JC-1. It was found thatTUBB4A expression level correlated with tumor grade, IDH1 status,1p/19q status, and poor survival in glioma patients. In addition, TUBB4A overexpression inhibited the proliferation, migration, and invasion of U251-MG and U87-MG, while increasing the degree of apoptosis. Notably, TUBB4A overexpression promotes ROS generation and MMP depolarization, and induces mitophagy through the PINK1/Parkin pathway. Interestingly, mitochondria-targeted ROS scavenger reversed the effect of TUBB4A overexpression on PINK1/Parkin expression and mitophagy, whereas mitophagy inhibitor did not affect ROS production. And the effect of TUBB4A overexpression on mitophagy and glioma progression was consistent with that of PINK1/Parkin agonist. In conclusion,TUBB4A is a molecular marker for predicting the prognosis of glioma patients and an effective target for inhibiting glioma progression by regulating ROS-PINK1/Parkin-mitophagy pathway.

First Report of Nigrospora oryzae Causing Leaf Spot on Peanut (Arachis hypogaea L.) in the Republic of Korea.

Peanut (Arachis hypogaea L.) is one of the most profitable upland crops, yielding 10,711 tonnes in an area of 4,062 ha in the Republic of Korea (Ministry of Agriculture, Food and Rural Affairs 2023). In September 2023, dark gray spots surrounded by yellow halos were observed on the peanut leaves over an area of 880 m2 at the National Institute of Crop Science (35°50'31.4"N 127°02'41.0"E), with a disease incidence up to 80%. Early symptoms appeared as small, brown, circular or irregular spots that enlarged and were surrounded by chlorotic halos. Leaf cuttings (5 mm x 5 mm) from five symptomatic plants were surface-sterilized with 70% EtOH for 1 min, followed by 1% NaClO for 1 min, and rinsed 3 times with sterile water. The pieces were placed on Potato Dextrose Agar (PDA) and incubated at 25 °C in the dark for 3 days. Three isolates obtained by single-spore isolation were designated as F23025, F23026, and F23027. Two isolates, F23025 and F23026 were deposited in the Korean Agricultural Culture Collection (https://genebank.rda.go.kr) under the accession numbers 410722 and 410723. Fungal colonies were initially white and turned sooty gray after 5 days. Conidia were unicellular, brown to black, and spherical or sub-spherical with 6.8 μm to 14.3 μm (mean = 11.1 μm ± 1.8, n = 50). The morphology of the three isolates was identical and showed the same characteristics as Nigrospora oryzae (Ellis 1971; Hudson 1963). For molecular identification, the Internal Transcribed Spacer (ITS) region (GenBank accession PP388306 and PP574448), beta tubulin (PP397027 and PP580108), and translation elongation factor 1- ɑ (PP397028 and PP580109) of isolates F23025 and F23026 were amplified and sequenced with primers of ITS5/ITS4, Bt2a/Bt2b, EF1-727F/EF2, respectively and showed high identity of 99.62% (530 bp/532 bp), 100% (384/384), and 99.79% (475/476) with N. oryzae strain LC2693 (GenBank accessions KX985994, KY019471, and KY019299, respectively). Multilocus sequence analysis showed isolates F23025 and F23026 were on the same clade with N. oryzae strain LC2693. To determine the pathogenicity to peanut, a conidial suspension (1 x 106 conidia/mL) was sprayed onto leaves of five 3-week-old plants 'Sewon' grown in pots, while sterile distilled water was sprayed onto two plants used as negative control. Sprayed plants were placed in a dew chamber at 25℃ for two days and grown in a growth chamber at 25℃ and 80% of relative humidity with a 16L:8D cycle. Two weeks later, dark spots with chlorotic halos appeared only on leaves sprayed with conidia, and no symptoms on leaves sprayed with sterile distilled water. The pathogenicity test was repeated three times, and each time the pathogen was re-isolated and identified by ITS sequence, thus fulfilling Koch's postulates. Nigrospora species are cosmopolitan, and some species have a wide host range as plant pathogens. Recently, two species of the genus Nigrospora, N. sphaerica and N. aurantiaca, were reported to cause peanut leaf blight in China (Liu et al. 2020; He et al. 2023). To the best of our knowledge, this is the first report of N. oryzae causing leaf spot to A. hypogaea L. in the Republic of Korea. As identifying new pathogens and registering fungicides to control them are important for the continued cultivation of peanut, this report will help in that endeavor.

Proteomic Dynamics of Multidrug Resistance Mechanisms in Lucena 1 Cell Line.

The Lucena 1 cell line, derived from the human chronic myeloid leukemia cell line K562 under selective pressure of vincristine supplementation, exhibits multidrug resistance (MDR). This study aims to explore and elucidate the underlying mechanisms driving MDR in the Lucena 1 cell line. A proteomic analysis comparing K562 and Lucena 1 revealed qualitative differences, with a focus on the ATP-dependent efflux pump, Translocase ABCB1, a key contributor to drug resistance. Tubulin analysis identified two unique isoforms, Tubulin beta 8B and alpha chain-like 3, exclusive to Lucena 1, potentially influencing resistance mechanisms. Additionally, the association of Rap1A and Krit1 in cytoskeletal regulation and the presence of STAT1, linked to the urea cycle and tumor development, offered insights into Lucena 1's distinctive biology. The increased expression of carbonic anhydrase I suggested a role in pH regulation. The discovery of COP9, a tumor suppressor targeting p53, further highlighted the Lucena 1 complex molecular landscape. This study offers new insights into the MDR phenotype and its multifactorial consequences in cellular pathways. Thus, unraveling the mechanisms of MDR holds promise for innovating cancer models and antitumor targeted strategies, since inhibiting the P-glycoprotein (P-gp)/ABCB1 protein is not always an effective approach given the associated treatment toxicity.

Abstract Or101: Phospho-Mimic Mutation in Beta Tubulin Suppresses Arrhythmias and Improves Cardiac Connexin 43 Localization in Duchenne Muscular Dystrophy Mice

Duchenne Muscular Dystrophy (DMD) is a severe X-linked neuromuscular disorder, causing progressive muscle degeneration and premature death due to heart and skeletal muscle dysfunction. It is caused by mutations in the dmd gene, leading to the absence of dystrophin, a crucial protein for muscle integrity. There is no cure for DMD. Its associated dystrophic cardiomyopathy is the primary cause of mortality by age 20. Connexin-43 (Cx43) is a gap junction protein responsible for electrical signaling in the heart. Cx43 localizes at the intercalated disc of the cardiomyocyte (CM), forming an intercellular channel with adjacent CMs; In DMD, Cx43 hemichannel remodels to the lateral side of the cardiomyocyte. Our previous research indicated that phospho-mimic Cx43 carrying serine to glutamic acid substitutions at the triplet regulatory site S325, S328, S330 in mdx hearts suppressed Cx43 lateralization and improved cardiac rhythm. The phospho-mimic Cx43 also reduced pathological hyperdensity and transverse directionality of microtubules in mdx cardiomyocytes. Colchicine (Colch) treatment, known to inhibit microtubule polymerization, also improved cardiac rhythm and Cx43 localization in mdx mice. To phenocopy the effect observed with Colch, we generated a mouse model with a beta tubulin mutation at serine 172, which was replaced by glutamic acid (S172E). We found reduced arrhythmias and improved localization of Cx43 in mdx hearts carrying the S172E phospho-mimic mutant tubulin by electrocardiography and immunofluorescent staining, respectively. Moreover, the proteomics data showed that the level of phospho-Cx43 was also upregulated in mdx hearts with S172E tubulin mutation. Further investigation will explore how this mutation impacts protein regulation and electrophysiology, and elucidate its mechanism of action in microtubule polymerization. These studies investigate a regulatory link between microtubule dynamics and localization of Cx43, potentially suggesting a novel therapeutic target for DMD-associated cardiomyopathy.

Deciphering the dynamic expression network of fiber elongation and the functional role of the GhTUB5 gene for fiber length in cotton based on an introgression population of upland cotton

IntroductionInterspecific introgression between Gossypium hirsutum and G. barbadense allows breeding cotton with outstanding fiber length (FL). However, the dynamic gene regulatory network of FL-related genes has not been characterized, and the functional mechanism through which the hub gene GhTUB5 mediates fiber elongation has yet to be determined. MethodsCoexpression analyses of 277 developing fiber transcriptomes integrated with QTL mapping using 250 introgression lines of different FL phenotypes were conducted to identify genes related to fiber elongation. The function of GhTUB5 was determined by ectopic expression of two TUB5 alleles in Arabidopsis and knockout of GhTUB5 in upland cotton. Yeast two-hybrid, split-luciferase and pull-down assays were conducted to screen for interacting proteins, and upstream genes were identified by yeast one-hybrid, dual-LUC and electrophoretic mobility shift assays. ResultsThe 32,612, 30,837 and 30,277 genes expressed at 5, 10 and 15 days postanthesis (dpa) were grouped into 19 distinct coexpression modules, and 988 genes in the MEblack module were enriched in the cell wall process and exhibited significant associations with FL. A total of 20 FL-QTLs were identified, each explaining 3.34–16.04 % of the phenotypic variance in the FL. Furthermore, several FL-QTLs contained 15 genes that were differentially expressed in the MEblack module including the tubulin beta gene (TUB5). Compared with the wild type, the overexpression of GhTUB5 and GbTUB5 in Arabidopsis suppressed root cell length but promoted cellulose synthesis. Knockout of GhTUB5 resulted in longer fiber lines. Protein-based experiments revealed that GhTUB5 interacts with GhZFP6. Additionally, GhTUB5 was directly activated by GhHD-ZIP7, a homeobox-leucine zipper transcription factor, and its paralogous gene was previously reported to mediate fiber elongation. ConclusionThis study opens a new avenue to dissect functional mechanism of cotton fiber elongation. Our findings provide some molecular details on how GhTUB5 mediates the FL phenotype in cotton.

LncRNA GAS5 modulates Schwann cell function and enhances facial nerve injury repair via the miR-138-5p/CXCL12 axis.

Facial nerve is an integral part of peripheral nerve. Schwann cells are important microglia involved in the repair and regulation of facial nerve injury. LncRNA growth arrest‑specific transcript 5 (GAS5) is involved in the behavioral regulation of Schwann cell and the regeneration of peripheral nervous system. However, there is little research about the effect of GAS5 on the repair of facial nerve injury (FNI) by regulating Schwann cells. This study aimed to investigate the role of GAS5 in Schwann cell function and FNI repair, focusing on the miR-138-5p/CXCL12 axis. Hematoxylin and eosin staining, Luxol fast blue staining, transmission electron microscope, and immunofluorescence (IF) experiments were used to verify the effect of GAS5 on FNI rats. Reverse transcription real-time polymerase chain reaction was performed to detect GAS5, miR-138-5p, and C-X-C motif chemokine ligand 12 (CXCL12) mRNA expression. IF staining was used to detect the inflorescence of S100 calcium binding protein B (S100β), SRY-box transcription factor 10 (SOX10), and tubulin beta 3 class III (β-Tubulin III). Glial fibrillary acidic protein (GFAP), nerve growth factor receptor (NGFR), S100β, brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and CXCL12 proteins were detected using western blot. The5-bromo-2'-deoxyuridine staining, Transwell, and flow cytometry assays were conducted to detect Schwann cell function. Dual-luciferase, RNA immunoprecipitation, and RNA pulldown assaywere used to identify the interaction among GAS5, miR-138-5p, and CXCL12. Results found thatGAS5 was downregulated in facial nerve tissues of FNI rats. Overexpressed GAS5 decreased facial grading, inhibited demyelination, and promoted proliferation, migration, and suppressed apoptosis of Schwann cells. Mechanistically, GAS5 was a sponge of miR-138-5p and positively regulated CXCL12 expression. GAS5 inhibition repressed CXCL12 expression and decreased cell proliferationand migration, increased apoptosis rate of Schwann cells by sponging miR-138-5p. In conclusion,overexpression of GAS5 accelerates facial nerve repair in FNI rats by regulating miR-138-5p/CXCL12 axis.

γ-tubulin complex controls the nucleation of tubulin-based structures in Apicomplexa.

Apicomplexan parasites rely on tubulin structures throughout their cell and life cycles, particularly in the polymerization of spindle microtubules to separate the replicated nucleus into daughter cells. Additionally, tubulin structures, including conoid and subpellicular microtubules, provide the necessary rigidity and structure for dissemination and host cell invasion. However, it is unclear whether these tubulin structures are nucleated via a highly conserved γ-tubulin complex or through a specific process unique to apicomplexans. This study demonstrates that Toxoplasma γ-tubulin is responsible for nucleating spindle microtubules, akin to higher eukaryotes, facilitating nucleus division in newly formed parasites. Interestingly, γ-tubulin colocalizes with nascent conoid and subpellicular microtubules during division, potentially nucleating these structures as well. Loss of γ-tubulin results in significant morphological defects due to impaired nucleus scission and the loss of conoid and subpellicular microtubule nucleation, crucial for parasite shape and rigidity. Additionally, the nucleation process of tubulin structures involves a concerted action of γ-tubulin and Gamma Tubulin Complex proteins (GCPs), recapitulating the localization and phenotype of γ-tubulin. This study also introduces new molecular markers for cytoskeletal structures and applies iterative expansion microscopy to reveal microtubule-based architecture in Cryptosporidium parvum sporozoites, further demonstrating the conserved localization and probable function of γ-tubulin in Cryptosporidium.

Evaluating some new benzimidazole derivative: Insilico docking studies for anthelmintic drug development

The Heterocyclic compounds used as medicinal agents in the field of study since long time. Benzo-fused heterocyclic nucleus benzimidazole founds significant class for new drug development. Molecular docking study is a technique in Computer Aided Drug Designing for studying ligand and protein interaction. The main motto of this work is to perform Preliminary screening using SAR studies, OSIRIS molecular property explorer, PASS Prediction Activity spectra and Rule of Five and to check the proposed benzimidazole compounds are potentially active as Anthelminthic agent by Docking studies with beta tubulin protein (PDB ID 1OJ0) using Auto dock 1.5.6 and VINA. Among all proposed benzimidazole derivatives, 4-((1H-benzimidazol-1-ylimino) methyl) phenol (5d) is the most effective and showed maximum binding energy (−7.4 K/Cal) when compared with standard drug albendazole (−7.1 K/Cal).

Response to Letter to the Editor: Tubulin beta is not the target of antineutrophil antibodies in primary sclerosing cholangitis.

Response to Letter to the Editor: Tubulin beta is not the target of antineutrophil antibodies in primary sclerosing cholangitis.

Tubulin beta 5 is not the target of antineutrophil antibodies in primary sclerosing cholangitis.

Tubulin beta 5 is not the target of antineutrophil antibodies in primary sclerosing cholangitis.

Toxicity of bisphenol A and p-nitrophenol on tomato plants: Morpho-physiological, ionomic profile, and antioxidants/defense-related gene expression studies.

Bisphenol A (BPA) and p-nitrophenol (PNP) are emerging contaminants of soils due to their wide presence in agricultural and industrial products. Thus, the present study aimed to integrate morpho-physiological, ionic homeostasis, and defense- and antioxidant-related genes in the response of tomato plants to BPA or PNP stress, an area of research that has been scarcely studied. In this work, increasing the levels of BPA and PNP in the soil intensified their drastic effects on the biomass and photosynthetic pigments of tomato plants. Moreover, BPA and PNP induced osmotic stress on tomato plants by reducing soluble sugars and soluble proteins relative to control. The soil contamination with BPA and PNP treatments caused a decline in the levels of macro- and micro-elements in the foliar tissues of tomatoes while simultaneously increasing the contents of non-essential micronutrients. The Fourier transform infrared analysis of the active components in tomato leaves revealed that BPA influenced the presence of certain functional groups, resulting in the absence of some functional groups, while on PNP treatment, there was a shift observed in certain functional groups compared to the control. At the molecular level, BPA and PNP induced an increase in the gene expression of polyphenol oxidase and peroxidase, with the exception of POD gene expression under BPA stress. The expression of the thaumatin-like protein gene increased at the highest level of PNP and a moderate level of BPA without any significant effect of both pollutants on the expression of the tubulin (TUB) gene. The comprehensive analysis of biochemical responses in tomato plants subjected to BPA and PNP stress illustrates valuable insights into the mechanisms underlying tolerance to these pollutants.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation.

Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0 ), TATA-binding protein (TBP ), GAPDH , actin beta (ACTB ), tubulin (TUB ), aminolevulinic acid synthase 1 (ALAS1 ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ ), eukaryotic translational elongation factor 1 alpha (EF1a ), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA ), and beta-2-microglobulin (B2M )] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms’ two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Selection of stable housekeeping genes for qPCR in biotic and abiotic stressed jute and their application in lignin pathway analysis

This is a comprehensive examination of housekeeping gene (HKG) expression in jute (Corchorus sp.), essential for reliable qRT-PCR studies. Jute is the most abundant and vital source of bast (phloem) fibres that have diverse domestic and industrial applications. The functional analysis of genes in jute is crucial for enhancing its commercial potential and value. We specifically focused on 24 HKGs, including nine previously recommended HKGs for jute. We focused the investigation on the only two commercially cultivated species of jute—C. capsularis and C. olitorius. The jute plants were subjected to nine abiotic stresses: osmotic, salinity, low temperature, high temperature, waterlogging, ABA stress hormone, glyphosate herbicide, and heavy metals (copper and cadmium), along with a biotic stress condition—Macrophomina phaseolina (fungal) infection. After rigorous analyses using the ΔCt method, BestKeeper, geNorm, NormFinder, and RefFinder, we identified ubiquitin1 (UBI) and beta-tubulin (BTUB) as the most stable HKGs for both the jute species under various stress conditions. The gene stability of UBI and BTUB was assessed in different jute cultivars, tissues, at different developmental stages, and PCR conditions. As a case study, we investigated eight genes involved in the jute lignin pathway in a low-lignin fibre-producing white jute mutant cultivar—dlpf—compared to its control cultivar, JRC212. Our findings revealed low expression of the three genes- CcCCoAoMT2, CcC4H1, and CcCAD7 in the dlpf cultivar, promising a potential for developing low-lignin jute varieties to meet long-standing industrial demands. The jute HKG expressions under various stress conditions establish a foundation for future research for crop improvement.