Search Results (34)

Background: Multiple myeloma (MM) is essentially an incurable cancer, but treatments with proteasome inhibitors are widely used clinically to extend patient survival. While the mechanisms of proteasome inhibition by Bortezomib are well known, the cellular responses to this proteotoxic stress that leads to sensitivity by MM are not fully elucidated. This study reports on the application of an emerging method to investigate proteostasis by proteomics. Methods: We utilized metabolic labeling with azidohomoalanine (AHA) in a MM cell line in combination with Bortezomib treatment. AHA labeling facilitates the selective isolation and identification of proteins for investigations of protein synthesis or protein degradation. Results: The data collected reveals significant changes in gene protein synthesis upon Bortezomib treatment, including protein neddylation. The data also reveals a global increase in protein degradation, which suggests the induction of an autophagy-related process. The resulting data collected reveals significant changes upon Bortezomib treatment in protein synthesis of genes, including protein neddylation, and protein degradation data reveals a global increase in protein degradation, suggesting an induction of an autophagy-related process. Subsequent cellular and proteomic analysis investigated the additional treatment of an autophagy inhibitor, hydroxychloroquine, in combination with Bortezomib treatment by label-free proteomics to further characterize the proteome-wide changes in these two proteotoxic stresses. Conclusions: AHA metabolic labeling proteomics to investigate protein synthesis and degradation enables novel complementary insights into complex cellular responses compared to that of traditional label-free proteomics. Full article

The hypothalamus is a key regulator of fundamental physiological processes and a site of adult neurogenesis. Allopregnanolone (ALLO) is a neurosteroid that mitigates the adverse effects of stress on the central nervous system and also affects neurogenesis. This study examined the effects of acute stress and ALLO administration (separately or in combination) into the third brain ventricle on the expression of neurotrophins and Trkβ receptor in distinct hypothalamic areas of sexually active female sheep. Expression of genes encoding brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and the Trkβ receptor was analyzed in the medial basal hypothalamus (MBH), arcuate nucleus (ARC), anterior hypothalamus (AHA), paraventricular nucleus (PVN), and preoptic area (POA). Acute stress stimulated the expression of neurotrophins (BDNF, NGF, and NT-3) in the ARC and PVN, while inhibitory effects predominated in the MBH, AHA and POA. ALLO alone mainly suppressed neurotrophins expression, while stimulatory effects were limited to the BDNF–Trkβ system in the ARC and Trkβ in the AHA. When combined with stress, ALLO either counteracted stress-induced increases in neurotrophins expression or produced no effect. The results demonstrate that acute stress can differentially modify neurotrophins mRNA expression in hypothalamic regions, activating neurotrophic activity in specific nuclei. The predominant inhibitory effect of ALLO on neurotrophin synthesis, particularly under conditions of acute stress, may help prevent excessive neuronal activation. Conversely, the upregulation of the BDNF-Trkβ system in the ARC indicates a positive relationship between this neurosteroid and hypothalamic adult neurogenesis. Full article

Ogura cytoplasmic male sterility (CMS) in Chinese cabbage (Brassica rapa) is characterized by complete pollen abortion, wherein stamens fail to produce viable pollen while pistils retain normal fertility. This maternally inherited trait is valuable for hybrid breeding. This study employed integrated analysis of miRNA, transcriptome, and degradome sequencing data aligned to the Chinese cabbage reference genome to elucidate the molecular function of bra-miR9569 in Ogura CMS pollen fertility and explore its associated pathways. Subsequently, a bra-miR9569 overexpression vector was constructed and transformed into Arabidopsis thaliana. Phenotypic characterization of transgenic Arabidopsis lines, combined with anther viability assessment and quantification of ATP content and reactive oxygen species (ROS) levels in Chinese cabbage, was performed to analyze the effects of bra-miR9569. Our findings demonstrate that mutation of the mitochondrial gene orf138 in Ogura CMS lines leads to upregulation of bra-miR9569. This microRNA negatively regulates the expression of the ATP-related gene AHA6, resulting in reduced H⁺-ATPase activity. The consequent energy deficiency triggers cellular content degradation, ultimately causing failure of pollen wall formation and pollen abortion. Full article

Soil salinization is a major constraint to global crop productivity, highlighting the need to identify salt tolerance genes and their molecular mechanisms. Here, we integrated mRNA and miRNA profile analyses to investigate the molecular basis of salt tolerance of an elite Brassica napus cultivar S268. Time-course RNA-seq analysis revealed dynamic transcriptional reprogramming under 215 mM NaCl stress, with 212 core genes significantly enriched in organic acid degradation and glyoxylate/dicarboxylate metabolism pathways. Combined with weighted gene co-expression network analysis (WGCNA) and RT-qPCR validation, five candidate genes (WRKY6, WRKY70, NHX1, AVP1, and NAC072) were identified as the regulators of salt tolerance in rapeseed. Haplotype analysis based on association mapping showed that NAC072, ABI5, and NHX1 exhibited two major haplotypes that were significantly associated with salt tolerance variation under salt stress in rapeseed. Integrated miRNA-mRNA analysis and RT-qPCR identified three regulatory miRNA-mRNA pairs (bna-miR160a/BnaA03.BAG1, novel-miR-126/BnaA08.TPS9, and novel-miR-70/BnaA07.AHA1) that might be involved in S268 salt tolerance. These results provide novel insights into the post-transcriptional regulation of salt tolerance in B. napus, offering potential targets for genetic improvement. Full article

Heat stress transcription factors (Hsfs) are crucial transcription factors (TFs) in plants, playing pivotal roles in responding to abiotic stresses. However, their specific functions in regulating heat stress responses in roses are not yet fully elucidated. Here, we cloned an Hsf gene, RhHsfA7, from the rose variety Rosa hybrid ‘Samantha’. This gene contains a coding sequence (CDS) of 1086 bp, encoding 361 amino acids. The RhHsfA7 protein has a molecular weight of 41.21 kDa, an isoelectric point of 5.41, and no signal peptide or transmembrane structure. Phylogenetic analyses revealed that RhHsfA7 is most closely related to AtHsfA7a, AtHsfA7b, and AtHsfA6b in Arabidopsis thaliana, and is phylogenetically closer to Rosaceae species compared to other species. The RhHsfA7 protein possesses conserved domains, including an oligomerization domain (OD), a nuclear localization signal (NLS), a DNA-binding domain (DBD), and a nuclear export signal (NES), as well as the HsfA subfamily-specific transcriptional activation domain (AHA). RhHsfA7 was localized in the nucleus and exhibited transcriptional activation activity. Expression analysis revealed that RhHsfA7 was highly expressed in roots and leaves, and its expression was heat-specific. In rose leaves, through silencing and transient overexpression experiments, we discovered that silencing RhHsfA7 resulted in heat sensitivity, whereas transient overexpression of RhHsfA7 increased heat tolerance. Collectively, our findings suggest that RhHsfA7 positively regulates tolerance to heat stress in roses. Full article

Iron (Fe) deficiency poses a major threat to pear (Pyrus spp.) fruit yield and quality. The Gretchen Hagen 3 (GH3) plays a vital part in plant stress responses. However, the GH3 gene family is yet to be characterized, and little focus has been given to the function of the GH3 gene in Fe deficiency responses. Here, we identified 15 GH3 proteins from the proteome of Chinese white pear (Pyrus bretschneideri) and analyzed their features using bioinformatics approaches. Structure domain and motif analyses showed that these PbrGH3s were relatively conserved, and phylogenetic investigation displayed that they were clustered into two groups (GH3 I and GH3 II). Meanwhile, cis-acting regulatory element searches of the corresponding promoters revealed that these PbrGH3s might be involved in ABA- and drought-mediated responses. Moreover, the analysis of gene expression patterns exhibited that most of the PbrGH3s were highly expressed in the calyxes, ovaries, and stems of pear plants, and some genes were significantly differentially expressed in normal and Fe-deficient pear leaves, especially for PbrGH3.5. Subsequently, the sequence of PbrGH3.5 was isolated from the pear, and the transgenic tomato plants with PbrGH3.5 overexpression (OE) were generated to investigate its role in Fe deficiency responses. It was found that the OE plants were more sensitive to Fe deficiency stress. Compared with wild-type (WT) plants, the rhizosphere acidification and ferric reductase activities were markedly weakened, and the capacity to scavenge reactive oxygen species was prominently impaired in OE plants under Fe starvation conditions. Moreover, the expressions of Fe-acquisition-associated genes, such as SlAHA4, SlFRO1, SlIRT1, and SlFER, were all greatly repressed in OE leaves under Fe depravation stress, and the free IAA level was dramatically reduced, while the conjugated IAA contents were notably escalated. Combined, our findings suggest that pear PbrGH3.5 negatively regulates Fe deficiency responses in tomato plants, and might help enrich the molecular basis of Fe deficiency responses in woody plants. Full article

Acetohydroxyacid synthase (AHAS) is a key enzyme in the first step of the branched-chain amino acid synthesis pathway, and the production of acetohydroxybutyrate from one molecule of 2-ketobutyric acid and one molecule of pyruvate. AHAS is inhibited by feedback from L-valine, L-leucine, and L-isoleucine, and the expression of ilvBN, the gene encoding AHAS, is regulated by all three branched-chain amino acids. A change in amino acids 20–22 on the regulatory subunit (M13 mutation) removes the feedback inhibition by valine. We cloned the gene encoding AHAS (ilvBN) into a vector and then transfected it into Escherichia coli BL21 for expression with targeted changes in amino acids 20–22 on the regulatory subunit, and then determined the activity of the mutated AHAS and its inhibitory effects on valine, isoleucine, and leucine. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Previous studies have suggested that the binding sites for the three branched-chain amino acids may be at the same variable center. We investigated the enzymatic properties of wild-type and mutant AHAS, modeled their crystal structures, and resolved the mechanism of feedback inhibition induced by mutant M13, which will be useful for continuing the modification of AHAS and the design of broad-spectrum herbicides. Full article

Acquired hemophilia A (AHA) is a bleeding disorder characterized by the immunological inhibition of factor VIII (FVIII) of the hemostatic pathway leading to hemorrhagic events. Different domains of FVIII are the target of autoantibodies (mainly immunoglobulin (Ig) G) leading to the deficiency of FVIII. Several factors have been associated with the activation of the auto-immunity towards FVIII. Emerging evidence implicates CD4+ T cell activation in mediating this autoimmune response, with their involvement like that observed in congenital hemophilia A. Several genes such as HLA II DRB*16, DQB1*0502, and CTLA-4 + 49 are responsible for the pathogenesis of AHA. Epigenetic modifications and mainly long-coding RNAS (lncRNAs) are potentially contributing to the pathogenesis of AHA. The treatment approach of AHA includes the management of acute bleeding events and the administration of immunosuppressive medications. This review aimed to summarize the published data on the genetics and epigenetics of AHA. The severity and the mortality of this disease are creating an emerging need for further research in the field of the genetics and epigenetics of acquired hemorrhagic disorder. Full article

Soil salinization poses a threat to the sustainability of agricultural production and has become a global issue. Cotton is an important cash crop and plays an important role in economic development. Salt stress has been harming the yield and quality of many crops, including cotton, for many years. In recent years, soil salinization has been increasing. It is crucial to study the mechanism of cotton salt tolerance and explore diversified materials and methods to alleviate the salt stress of cotton for the development of the cotton industry. Nanoparticles (NPs) are an effective means to alleviate salt stress. In this study, zinc oxide NPs (ZnO NPs) were sprayed on cotton leaves with the aim of investigating the intrinsic mechanism of NPs to alleviate salt stress in cotton. The results show that the foliar spraying of ZnO NPs significantly alleviated the negative effects of salt stress on hydroponic cotton seedlings, including the improvement of above-ground and root dry and fresh weight, leaf area, seedling height, and stem diameter. In addition, ZnO NPs can significantly improve the salt-induced oxidative stress by reducing the levels of MDA, H₂O₂, and O₂⁻ and increasing the activities of major antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Furthermore, RNA-seq showed that the foliar spraying of ZnO NPs could induce the expressions of CNGC, NHX2, AHA3, HAK17, and other genes, and reduce the expression of SKOR, combined with the CBL-CIPK pathway, which alleviated the toxic effect of excessive Na⁺ and reduced the loss of excessive K⁺ so that the Na⁺/K⁺ ratio was stabilized. In summary, our results indicate that the foliar application of ZnO NPs can alleviate high salt stress in cotton by adjusting the Na⁺/K⁺ ratio and regulating antioxidative ability. This provides a new strategy for alleviating the salt stress of cotton and other crops, which is conducive to the development of agriculture. Full article

As the only aquatic lineage of Pteridaceae, Parkerioideae is distinct from many xeric-adapted species of the family and consists of the freshwater Ceratopteris species and the only mangrove ferns from the genus Acrostichum. Previous studies have shown that whole genome duplication (WGD) has occurred in Parkerioideae at least once and may have played a role in their adaptive evolution; however, more in-depth research regarding this is still required. In this study, comparative and evolutionary transcriptomics analyses were carried out to identify WGDs and explore their roles in the environmental adaptation of Parkerioideae. Three putative WGD events were identified within Parkerioideae, two of which were specific to Ceratopteris and Acrostichum, respectively. The functional enrichment analysis indicated that the lineage-specific WGD events have played a role in the adaptation of Parkerioideae to the low oxygen concentrations of aquatic habitats, as well as different aquatic environments of Ceratopteris and Acrostichum, such as the adaptation of Ceratopteris to reduced light levels and the adaptation of Acrostichum to high salinity. Positive selection analysis further provided evidence that the putative WGD events may have facilitated the adaptation of Parkerioideae to changes in habitat. Moreover, the gene family analysis indicated that the plasma membrane H⁺-ATPase (AHA), vacuolar H⁺-ATPase (VHA), and suppressor of K⁺ transport growth defect 1 (SKD1) may have been involved in the high salinity adaptation of Acrostichum. Our study provides new insights into the evolution and adaptations of Parkerioideae in different aquatic environments. Full article

The transcriptional regulators of the MarR family play an important role in diverse bacterial physiologic functions, whereas their effect and intrinsic regulatory mechanism on the aquatic pathogenic bacterium Aeromonas hydrophila are, clearly, still unknown. In this study, we firstly constructed a deletion strain of AHA_2124 (ΔAHA_2124) of a MarR family transcriptional regulator in Aeromonas hydrophila ATCC 7966 (wild type), and found that the deletion of AHA_2124 caused significantly enhanced hemolytic activity, extracellular protease activity, and motility when compared with the wild type. The differentially abundant proteins (DAPs) were compared by using data-independent acquisition (DIA), based on a quantitative proteomics technology, between the ΔAHA_2124 strain and wild type, and there were 178 DAPs including 80 proteins up-regulated and 98 proteins down-regulated. The bioinformatics analysis showed that the deletion of gene AHA_2124 led to some changes in the abundance of proteins related to multiple biological processes, such as translation, peptide transport, and oxidation and reduction. These results provided a theoretical basis for better exploring the regulatory mechanism of the MarR family transcriptional regulators of Aeromonas hydrophila on bacterial physiological functions. Full article

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by the presence of autoantibodies against factor VIII (FVIII). As with other autoimmune diseases, its etiology is complex and its genetic basis is unknown. The aim of this study was to identify the immunogenetic background that predisposes individuals to AHA. HLA and KIR gene clusters, as well as KLRK1, were sequenced using next-generation sequencing in 49 AHA patients. Associations between candidate genes involved in innate and adaptive immune responses and AHA were addressed by comparing the alleles, genotypes, haplotypes, and gene frequencies in the AHA cohort with those in the donors’ samples or Spanish population cohort. Two genes of the HLA cluster, as well as rs1049174 in KLRK1, which tags the natural killer (NK) cytotoxic activity haplotype, were found to be linked to AHA. Specifically, A*03:01 (p = 0.024; odds ratio (OR) = 0.26[0.06–0.85]) and DRB1*13:03 (p = 6.8 × 10³, OR = 7.56[1.64–51.40]), as well as rs1049174 (p = 0.012), were significantly associated with AHA. In addition, two AHA patients were found to carry one copy each of the low-frequency allele DQB1*03:09 (n_allele = 2, 2.04%), which was completely absent in the donors. To the best of our knowledge, this is the first time that the involvement of these specific alleles in the predisposition to AHA has been proposed. Further molecular and functional studies will be needed to unravel their specific contributions. We believe our findings expand the current knowledge on the genetic factors involved in susceptibility to AHA, which will contribute to improving the diagnosis and prognosis of AHA patients. Full article

The aim of this study was to investigate the possible genotypic differences between commensal Pasteurella multocida isolates from apparently healthy animals (AHA) at the time of entry to feedlots and those from BRD-affected animals (BRD-AA). A total of 20 batches of beef calves in seven feedlots were followed-up during the fattening period. P. multocida was isolated from 28.1% of AHA and 22.9% of BRD-AA. All isolates belonged to the A: L3 genotype. Most isolates from clinical cases (81.0%) grouped into a PFGE cluster were significantly associated with BRD cases (OR, 24.9; 95% CI, 6.4–96.2). The whole genomes of 14 isolates representative of the pulsotypes most frequently detected in BRD-AA and AHA were sequenced and compared with 53 bovine genomes belonging to the identified ST13, ST79, and ST80 genotypes for a global comparison. No differences were found in the virulence-associated gene content between sequence types (STs) globally or between BRD-AA and AHA isolates in this study. Significantly, ST79 isolates harbored ARGs, conferring resistance to different antimicrobials, including macrolides and tetracyclines, which are commonly used for the treatment of BRD. Two Spanish ST79 isolates carried an ICE highly similar to ICE Tn7407, which was recently detected in Germany, suggesting that ST79 P. multocida isolates in Europe and North America may be associated with different ICEs. Full article

Atriplex spp. (saltbush) is known to survive extremely harsh environmental stresses such as salinity and drought. It mitigates such conditions based on specialized physiological and biochemical characteristics. Dehydrin genes (DHNs) are considered major players in this adaptation. In this study, a novel DHN gene from Azrak (Jordan) saltbush was characterized along with other Atriplex species from diverse habitats. Intronless DHN-expressed sequence tags (495–761 bp) were successfully cloned and sequenced. Saltbush dehydrins contain one S-segment followed by three K-segments: an arrangement called SK3-type. Two substantial insertions were detected including three copies of the K2-segemnet in A. canescens. New motif variants other than the six-serine standard were evident in the S-segment. AhaDHN1 (A. halimus) has a cysteine residue (SSCSSS), while AgaDHN1 (A. gardneri var. utahensis) has an isoleucine residue (SISSSS). In contrast to the conserved K1-segment, both the K2- and K3-segment showed several substitutions, particularly in AnuDHN1 (A. nummularia). In addition, a parsimony phylogenetic tree based on homologs from related genera was constructed. The phylogenetic tree resolved DHNs for all of the investigated Atriplex species in a superclade with an 85% bootstrap value. Nonetheless, the DHN isolated from Azraq saltbush was uniquely subclustred with a related genera Halimione portulacoides. The characterized DHNs revealed tremendous diversification among the Atriplex species, which opens a new venue for their functional analysis. Full article

This study was conducted to develop a novel herbicide resistance soybean using ethyl methanesulfonate (EMS) mutagen. In this study, 0.1% of EMS mutagen was applied to the soybean [Glycine max (L.) cv Arısoy] seeds. A single resistant mutant was selected in the M₂ population evaluated under field and greenhouse conditions. The AHAS gene regions of the herbicide-resistant mutant progeny were mapped, and the nucleotide changes were defined conferring herbicide resistance. The sequence analysis of the AHAS gene indicated that three nucleotide substitutions were detected such as 407 (C/T), 532 (C/T), and 1790 (C/T). According to the AHAS gene protein sequence of Arabidopsis thaliana, Ala155Val, Pro197Ser, and Thr616Met amino acid alterations were found in the progeny of the resistant mutant. Pro197Ser alteration was common in all the progeny, while the others were diverse. The wild-type and the mutant plants were compared for seed yield, number of pods per plant, stem height to the first pod, 1000-seed weight, and physiological maturity days for two subsequent years. No statistical difference was found between the mutant and wild types with respect to seed yield and its components. The agronomic data indicated that EMS provided target-site resistance to sulfonylureas (SU) with no tradeoff between yield components and resistance. Full article