Relative Gene Expression Analysis Research Articles

Chitosan nanoparticles of imatinib mesylate coated with TPGS for the treatment of colon cancer: In-vivo & in-vitro studies

The study aimed to develop and evaluate chitosan-based nanoparticles coated with TPGS for the targeted delivery of imatinib mesylate to colon cancer cells. Particle size and zeta potential analysis were within the acceptable range for targeting colon cancer. CS-IMT-TPGS-NPs had a significant positive zeta potential of 30.4 mV, suggesting improved cellular intake. FE-SEM and TEM demonstrated that the nanoparticles appeared spherical, smooth, and did not aggregate, with a visible TPGS coating. XRD confirmed that crystalline imatinib transitioned to an amorphous state during nano formulation. In-vitro tests on HCT-116 cells demonstrated that CS-IMT-TPGS-NPs outperformed free IMT regarding cytotoxicity, apoptosis induction, cellular uptake, and cell migration inhibition. Additionally, the nanoparticles were examined in vitro using mitochondrial membrane potential, DNA fragmentation, GAPDH relative gene expression, ROS estimation, and cell cycle analysis. The effect of therapy on expected colon-associated bacterial strains was also investigated. The biocompatibility of nanoparticles was assessed by hemolysis and platelet aggregation experiments. The anti-inflammatory impact was determined using the HET-CAM test. Non-Fickian diffusion at pH 5.5 resulted in sustained in-vitro drug release, with no initial burst. In-vivo investigations using albino Wistar rats suggest pharmacokinetic properties for produced nanoparticles, whereas histopathological examinations assess acute toxicity.

Mining and expression analysis of color related genes in Bougainvillea glabra bracts based on transcriptome sequencing.

Bract coloration is one of the key ornamental traits in Bougainvillea, yet research has predominantly focused on phenotypic color traits and pigment composition, with limited understanding of the molecular mechanisms underlying color formation. This gap hinders the improvement and innovation in bract coloration. To elucidate the regulatory mechanisms of bract coloration in Bougainvillea and to enhance the utilization of its germplasm resources, this study employed the Illumina Novaseq 6000 sequencing platform to conduct transcriptomic sequencing on 21 samples of bracts exhibiting seven distinct phenotypes. Comparative analysis against Nr, Pfam, EggNOG, GO, and KEGG databases annotated 90,279 unigenes. The highest annotation rates were achieved with the Nr (40.13%), GO (30.44%), and EggNOG (25.64%) databases. Among the species annotated, Beta vulgaris (20.08%) and Chenopodium quinoa (14.58%) shared the highest homology with Bougainvillea bract transcriptomes. WGCNA analysis identified 12 positively correlated tissue-specific modules, of which 2 are related to bract color formation. By comparing transcriptome data and genes within these specific modules against the KEGG database, a total of 321 unigenes associated with bract color formation in Bougainvillea were discovered. Among these, 220 unigenes are involved in anthocyanin synthesis, 43 unigenes are involved in betalain synthesis, 23 unigenes are annotated as Chlorophyll a-b binding protein genes, and 35 unigenes participate in carotenoid synthesis. Quantitative real-time PCR (qRT-PCR) validation of 16 differentially expressed genes (DEGs) including PAL2, CHS1, ANS, BZ1, 6GT, CDOPA5GT, ANR, CHS2, and DOPA, revealed significant expression differences among magenta, yellow, white, and cherry-colored bracts, suggesting their potential as candidate genes for bract color development. This study not only enriches the transcriptomic data of Bougainvillea but also identifies genes associated with bract coloration, providing a valuable theoretical basis for future gene cloning, genetic engineering, and breeding efforts in Bougainvillea.

PSVII-19 Effects of silage particle size on beef quality and muscle lipogenesis of finishing Nellore heifers

Abstract The objective of this study was to evaluate the effect of corn silage particle size on beef quality and expression of gluconeogenic and lipogenic genes in feedlot-finished Nellore heifers. Nellore heifers (n = 94), with an initial body weight (BW) of 249.71 ± 34.62, were allotted into 32 pens (three animals per pen) in a completely randomized design, with 2 treatments and 16 repetitions. The treatments were corn silage with a particle size of 13 mm (short) or 24 mm (long). The feeding period lasted 101 d, with the first 15 d for diet adaptation. After slaughter, samples of the longissimus thoracis muscle (LT) from the left side of the half carcass and liver were taken for beef quality and gene expression analyses. The chemical composition of the beef was analyzed using near infrared according to the AOAC method 2007-04, using the FoodScanTM equipment (AOAC method: 2007-04; FOSS, Hillerod, Denmark). RT-qPCR analyses of each gene investigated were carried out using cDNA from 16 biological replicates per treatment (1 heifer chosen randomly in each pen), with each biological replicate subjected to 2 technical replicates. Relative gene expression analyses were calculated using the delta delta ct method and the reference genes used were ACTB and GAPDH. The normality of all experimental data was checked using the Shapiro-Wilk test in SAS software. When the data did not present a normal distribution, transformation was performed using PROC RANK. The model included particle size as a fixed effect and pen as a random effect. There was no treatment effect on collagen, moisture, and mineral content (P > 0.10), but muscles from heifers fed short particle sizes tended to have greater protein content (23.3 versus 22.9; P = 0.098), while muscles from heifers fed with long particles had greater intramuscular fat content (2.62 versus 3.28; P = 0.05). For liver gene expression, heifers fed short particle sizes tended to have greater expression of the PC gene (1.00 versus 0.82; P = 0.06), while PEPCK2 had higher expression in heifers fed long particles (1.00 versus 1.18; P = 0.04). In addition, the long particle size of corn silage affected the expression of PPARA (P = 0.001), and there was no effect of diets (P > 0.05) on PPARG and SREBF1 expressions (Table 1). Expression of LPL, FABP4, SCD1, CPT2 (P < 0.001), and ACACA (P = 0.017) genes increased in the muscle of heifers fed long particle size. Therefore, it is concluded that the use of corn silage with long particle size in feedlot diets has the potential to increase the expression of genes involved in lipogenesis and then increase intramuscular fat in Nellore heifers.

Prognostic significance of miR 499 expression and Helicobacter pylori infection in malignant lesions of gallbladder cancer: a clinicopathological study

BackgroundGallbladder cancer (GBC) is an infrequent type of malignant neoplasm worldwide. There are a number of risk factors that increase a person's likelihood of developing GBC. Gallbladder inflammatory (GID) diseases including cholelithiasis increase the risk of GBC, and this is further complicated by the fact that Helicobacter pylori (H. pylori) infection is extremely common in gastrointestinal tract in India. Since both miR 499 and H. pylori infection are found to be linked with tumor progression and metastasis, therefore there is a possibility that H. pylori might be involved in inflammation via dysregulation of miR 499. The study was designed to investigate the association of miR 499 expressions with H. pylori infection and their correlation with clinicopathological parameters of GBC.Material and methodsThe hundred three tissue samples used in this study are categorized into GID (n = 55) and GBC (n = 48). The expression of miR-499 was examined by using the Livak method for relative gene expression analysis. The presence/absence of H. pylori infection was examined by RT-PCR (Liferiver Helicobacter pylori RT-PCR Kit).ResultsHelicobacter pylori infection and GBC/GID cases were not significantly correlated. Decreased expression of miR 499 was observed in GBC (1.6 fold) as compared to GID patients (P < 0.0001). Low miR 499 expression was found to significantly correlate with tumor differentiation (P = 0.017), advanced staging (P = 0.004) and liver metastasis (P = 0.036). Multivariate regression analysis showed significant association of overall survival with low miR 499 expressions.ConclusionmiR 499 may be considered as a useful prognostic biomarker in GBC progression.

Comparative genome-wide identification and characterization of SET domain-containing and JmjC domain-containing proteins in piroplasms

BackgroundSET domain-containing histone lysine methyltransferases (HKMTs) and JmjC domain-containing histone demethylases (JHDMs) are essential for maintaining dynamic changes in histone methylation across parasite development and infection. However, information on the HKMTs and JHDMs in human pathogenic piroplasms, such as Babesia duncani and Babesia microti, and in veterinary important pathogens, including Babesia bigemina, Babesia bovis, Theileria annulata and Theileria parva, is limited.ResultsA total of 38 putative KMTs and eight JHDMs were identified using a comparative genomics approach. Phylogenetic analysis revealed that the putative KMTs can be divided into eight subgroups, while the JHDMs belong to the JARID subfamily, except for BdJmjC1 (BdWA1_000016) and TpJmjC1 (Tp Muguga_02g00471) which cluster with JmjC domain only subfamily members. The motifs of SET and JmjC domains are highly conserved among piroplasm species. Interspecies collinearity analysis provided insight into the evolutionary duplication events of some SET domain and JmjC domain gene families. Moreover, relative gene expression analysis by RT‒qPCR demonstrated that the putative KMT and JHDM gene families were differentially expressed in different intraerythrocytic developmental stages of B. duncani, suggesting their role in Apicomplexa parasite development.ConclusionsOur study provides a theoretical foundation and guidance for understanding the basic characteristics of several important piroplasm KMT and JHDM families and their biological roles in parasite differentiation.

Integrated analysis of yield response and early stage biochemical, molecular, and gene expression profiles of pre-breeding rice lines under water deficit stress

Breeding high yielding water-deficit tolerant rice is considered a primary goal for achieving the objectives of the sustainable development goals, 2030. However, evaluating the performance of the pre-breeding-promising parental-lines for water deficit tolerance prior to their incorporation in the breeding program is crucial for the success of the breeding programs. The aim of the current investigation is to assess the performance of a set of pre-breeding lines compared with their parents. To achieve this goal a set of 7 pre-breeding rice lines along with their parents (5 genotypes) were field evaluated under well-irrigated and water-stress conditions. Water stress was applied by flush irrigation every 12 days without keeping standing water after irrigation. Based on the field evaluation results, a pre-breeding line was selected to conduct physiological and expression analysis of drought related genes at the green house. Furthermore, a greenhouse trial was conducted in pots, where the genotypes were grown under well and stress irrigation conditions at seedling stage for physiological analysis and expression profiling of the genotypes. Results indicated that the pre-breeding lines which were high yielding under water shortage stress showed low drought susceptibility index. Those lines exhibited high proline, SOD, TSS content along with low levels of MDA content in their leaves. Moreover, the genotypes grain yield positively correlated with proline, SOD, TSS content in their leaves. The SSR markers RM22, RM525, RM324 and RM3805 were able to discriminate the tolerant parents from the sensitive one. Expression levels of the tested drought responsive genes revealed the upregulation of OsLEA3, OsAPX2, OsNAC1, OSDREB2A, OsDREB1C, OsZIP23, OsP5CS, OsAHL1 and OsCATA genes in response to water deficit stress as compared to their expression under normal irrigated condition. Taken together among the tested pre-breeding lines the RBL112 pre-breeding line is high yielding under water-deficit and could be used as donor for high yielding genes in the breeding for water deficit resistance. This investigation withdraws attention to evaluate the promising pre-breeding lines before their incorporation in the water deficit stress breeding program.

Inflammatory responses in Atlantic lumpfish (Cyclopterus lumpus L.) after intraperitoneal injection of a vaccine against Aeromonas salmonicida and Vibrio salmonicida at different water temperatures.

Studying inflammatory responses induced by vaccination can contribute to a more detailed understanding of underlying immune mechanisms in lumpfish (Cyclopterus lumpus). Tissue samples from lumpfish intraperitoneally immunized with a divalent oil-adjuvanted vaccine (Aeromonas salmonicida and Vibrio salmonicida) at water temperatures of 5, 10, and 15°C were collected at 630 day degrees and 18 weeks post injection. The relative amount of secretory and membrane-bound immunoglobulin M (IgM) gene transcripts in the head kidney was determined by qPCR. Vaccine-induced inflammatory lesions were assessed on histological sections of abdominal pancreatic/intestinal tissue from vaccinated fish in all three temperature groups. Inflammatory cells forming dense aggregations in lesions showed proliferative activity, many of which were identified as eosinophilic-granulocyte-like cells. IgM+ cells were scattered in inflammatory tissue dominated by connective tissue, showing no difference in numbers between lesions from fish vaccinated at 5, 10, and 15°C. Relative gene expression analysis of secretory and membrane-bound IgM revealed low overall expression in the head kidney of vaccinated fish at both 630 day-degrees and 18 weeks post injection. The results of this study indicate that the vaccine stimulated prolonged local inflammatory responses at the injection site, which were not influenced by temperature.

Analysis of Relative Gene Expression of some Brain-Pituitary-Gonad Axis Genes of the Ripe Wild and Captive Female Liza ramada

Analysis of Relative Gene Expression of some Brain-Pituitary-Gonad Axis Genes of the Ripe Wild and Captive Female Liza ramada

Zebrafish as a model for the study of wound healing in hyperglycemia

Background: For preclinical studies of diabetic wound therapy, zebrafish have many orthologous signalling pathways that play essential roles in wound healing and regeneration. Objectives: This study compares the expression of four essential wound-healing genes and caudal fin regeneration in hyperglycemic zebrafish (Danio rerio) to normal zebrafish. Methods: Hyperglycemia was induced by using the intraperitoneal injection method with 350 mg/BW streptozotocin on days one, three, and five. The regeneration of the zebrafish's caudal fin was observed on day 5 after amputation using a stereomicroscope, followed by sampling of the blastema to analyse gene expression. Caudal fin regeneration was analysed using Zeiss Zen 3.3 blue documentation; blood glucose levels were measured using a glucometer; and relative gene expression analysis of sonic hedgehog (shh), insulin-like growth factor 2a (igf2a), bone morphogenetic protein 2b (bmp2b), collagen 1a2 (col1a2) was performed by qRT-PCR method. Results: The glucose level in the hyperglycemia group was 203.2 mg/dL, and that in the normal group was 59.5 mg/dL. The authors found that igf2a, shh, bmp2b, and col1a2 expression were all downregulated in the hyperglycemia group and decreased in caudal fin regeneration. Conclusion: This novel adult zebrafish model of hyperglycemia significantly impairs gene expression and caudal fin regeneration.

Expression analysis of necroptosis related genes and lncRNAs in patients with pituitary neuroendocrine tumors

Necroptosis can either be the cause of tumorigenesis or it can impede its process. Recently, it has been proved that long non-coding RNAs (lncRNAs) have different crucial roles at cellular level, especially on cell death. Regarding the important role of necroptosis and lncRNAs in the pathogenesis of different cancers, especially pituitary adenomas (PAs), we assessed expression levels of two necroptosis related genes, namely TRADD and BIRC2, in addition to three related lncRNAs, namely FLVCR1-DT, MAGI2-AS3, and NEAT1 in PAs compared with adjacent normal tissues (ANTs). TRADD had no significant difference between two groups; however, BIRC2, FLVCR1-DT, MAGI2-AS3, and NEAT1 were upregulated in PAs compared to ANTs (Expression ratios [95% CI] = 2.3 [1.47–3.6], 2.13 [1.02–4.44], 3.01 [1.76–5.16] and 2.47 [1.37–4.45], respectively). When taking into account different types of PAs, significant upregulation of BIRC2, MAGI2-AS3 and NEAT1 was recorded in non-functioning PAs compared with corresponding ANTs (Expression ratios [95% CI] =1.9 [1.04–3.43], 2.69 [1.26–5.72] and 2.22 [0.98–5.01], respectively). Additionally, higher levels of BIRC2 were associated with higher flow of CSF (P value=0.048). Moreover, higher Knosp classified tumors had lower levels of BIRC2 (P value=0.001). Finally, lower levels of MAGI2-AS3 were associated with larger tumor size (P value=0.006). NEAT1 expression was correlated with FLVCR1-DT and TRADD. TRADD expression was correlated with FLVCR1-DT. Additional correlation was observed between expression of BIRC2 and MAGI2-AS3. In sum, this study provides evidence that dysregulated levels of studied genes could contribute to the pathogenesis of pituitary tumors.

Molecular characterization of Lernathropus kroyeri from intensive aquaculture and pathophysiology of infested sea bass

The copepod Lernathropus kroyeri constitutes one of the major parasites for the Mediterranean aquaculture, infesting the sea bass Dicentrarchus labrax causing thus disruptions of growth performance and occasionally mortalities. Despite the large spread and the high frequency of this parasite in mariculture farms of Eastern Mediterranean, L. kroyeri genetic profile from aquaculture as well as the pathophysiological response of D. labrax have not been studied so far. Keeping this in mind, in the present study we investigated the L. kroyeri infestation on D. labrax from two farms in Greece, examining both healthy and heavy parasitized individuals. Assays included histopathology, phylogenetic reconstruction of the parasite and physiological response of the fish by the means of antioxidant, inflammatory metabolic and stress related gene expression analysis at both mRNA and protein levels. Genetic analysis indicated that L. kroyeri composes a monophyletic group, highly phylogenetically distant from other congeneric groups. Heavy infested D. labrax witnessed a significantly increased immune response that further led to oxidative stress and metabolic alterations. Overall, our results demonstrate the, seasonally independent, high infestation of this parasitic copepods, which continue to affect Mediterranean intensive aquaculture systems.

The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 μg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

Relative gene expression analysis of catalase in environmental RNA from Japanese medaka exposed to toxic chemicals

AbstractEnvironmental RNA (eRNA) has the potential as a non‐invasive tool for assessing the physiological status of macro‐organisms, yet the quantitative method for relative gene expression analysis is still underexplored. To bridge this gap, this study introduces a relative quantification method for eRNA, employing quantitative PCR (qPCR) to evaluate the expression of the antioxidant gene, catalase (cat), in the Japanese medaka Oryzias latipes exposed to two toxic chemicals for 96 h: chlorpyrifos (CPS) and carbamazepine. Our results showed that, in eRNA, genes frequently used as a reference for tissue or cells in conventional expression analysis correlated with each other. Also, one of them (elfa) exhibited less variability, showing its potential suitability as a reference gene in eRNA analyses. Additionally, cat expression levels in eRNA increased with increasing CPS concentrations, in a concentration‐response manner. These results suggest the promising potential of qPCR applications for eRNA in the monitoring of an organism's health and response to environmental changes. However, we observed disparities in gene expression levels of cat and beta‐actin (actb) between eRNA and whole fish body, indicating eRNA might have a biased origin. Further research is needed to uncover the origin of eRNA and determine the limitations and applicability domain of eRNA analysis.

Interferon-β deficiency alters brain response to chronic HIV-1 envelope protein exposure in a transgenic model of NeuroHIV

Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and β. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNβ (IFNβKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNβ but in a sex-dependent fashion. Notably, in cerebral cortex of IFNβKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNβKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNβ-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNβ-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNβKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNβ on multiple components with more pronounced changes in IFNβKO females. In contrast, the effects of IFNβKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNβ impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNβ plays a vital role in maintaining neuronal homeostasis and memory function.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Unveiling the Cell Wall-Targeting Mechanisms and Multifaceted Virulence Modulation by a Eugenol Glycoconjugate against Aspergillus fumigatus: Insights from in vitro and in ovo Studies.

The primary objective of this study was to elucidate the putative cell wall-associated targets of compound 6i, a glycoconjugate of eugenol, in Aspergillus fumigatus, while also evaluating its toxicity and assessing histopathologic alterations in the liver, heart, and kidney of compound 6i-treated embryos using an in ovo model. To achieve this aim, compound 6i was synthesized, and a series of biochemical assays were performed to determine its impact on the fungal cell wall. Additionally, qRT-PCR and LC-MS/MS analyses were conducted to investigate changes in gene and protein expression profiles associated with melanin biosynthesis, conidiation, siderophore production, transcriptional regulation of β-glucan biosynthesis, and calcineurin activity in A. fumigatus. The experimental findings revealed that compound 6i exhibited notable antifungal activity against A. fumigatus by perturbing cell wall integrity, hindering ergosterol, glucan, and chitin biosynthesis, and inhibiting catalase production. Moreover, relative gene expression and proteomic analyses demonstrated that compound 6i exerted both down-regulatory and up-regulatory effects on several crucial genes and proteins involved in the aforementioned fungal processes. Furthermore, increased expression of oxidative stress-related proteins was observed in the presence of compound 6i. Notably, the glycoconjugate of eugenol did not elicit cytotoxicity in the liver, heart, and kidney of chick embryos. The current investigation elucidated the multifaceted mechanisms by which compound 6i exerts its antifungal effects against A. fumigatus, primarily through targeting cell wall components and signaling pathways. These findings underscore the potential of the eugenol glycoconjugate as a promising antifungal candidate, warranting further exploration and development for combating A. fumigatus infections.

A protocol for identifying universal reference genes within a genus based on RNA-Seq data: a case study of poplar stem gene expression.

Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) plays a crucial role in relative gene expression analysis, and accurate normalization relies on suitable reference genes (RGs). In this study, a pipeline for identifying candidate RGs from publicly available stem-related RNA-Seq data of different Populus species under various developmental and abiotic stress conditions is presented. DESeq2's median of ratios yielded the smallest coefficient of variance (CV) values in a total of 292 RNA-Seq samples and was therefore chosen as the method for sample normalization. A total of 541 stably expressed genes were retrieved based on the CV values with a cutoff of 0.3. Universal gene-specific primer pairs were designed based on the consensus sequences of the orthologous genes of each Populus RG candidate. The expression levels of 12 candidate RGs and six reported RGs in stems under different abiotic stress conditions or in different Populus species were assessed by RT-qPCR. The expression stability of selected genes was further evaluated using ΔCt, geNorm, NormFinder, and BestKeeper. All candidate RGs were stably expressed in different experiments and conditions in Populus. A test dataset containing 117 RNA-Seq samples was then used to confirm the expression stability, six candidate RGs and three reported RGs met the requirement of CV ≤ 0.3. In summary, this study was to propose a systematic and optimized protocol for the identification of constitutively and stably expressed genes based on RNA-Seq data, and Potri.001G349400 (CNOT2) was identified as the best candidate RG suitable for gene expression studies in poplar stems.

Exploring OsHAK Transporters for the Salt Responsiveness in Rice

Environmental stresses are the major constraint in crop growth and productivity, particularly in rice. Improving rice against major abiotic stressors such as salinity is one of the major thrust areas. In this direction, the trait of sodium exclusion was the primary focus among the rice breeders to enhance salt tolerance. Along similar lines, increased uptake of potassium, the counter ion of Na+, through high-affinity potassium transporters (HAK) assumes significance in the context of maintaining a favorable Na/K ratio, a low Na/K is an indicator of salt tolerance. The OsHAK family in rice includes 27 members and an insight into the evolution, structure, and expression through in silico approaches was attempted. Phylogenetic analysis exhibited four distinct groupings of the OsHAK family, and functional motif analysis revealed a characteristic consensus sequence among the OsHAK transporters. Relative gene expression analysis based on published microarray data put forth the differential regulation of OsHAK3 X2, OsHAK8, OsHAK14(X2), OsHAK15 and OsHAK26 between salt-tolerant landrace Pokkali and susceptible cultivar IR29 and their tissue-specific expression profiles predicted. This study narrowed down a set of putative OsHAKs from the larger family for their role in enhancing cellular potassium under salt stress thus paving the way for crop improvement.

The mechanism of melatonin promotion on cucumber seedling growth at different nitrogen levels

The supply level of exogenous nitrogen has a very important influence on the growth and development of cucumber. Insufficient or excessive nitrogen application will lead to metabolic disorders in the body and affect the formation of yield. Therefore, it is of great scientific and practical significance to explore the corresponding mitigation measures. Melatonin (MT) is a multi-regulatory molecule with pleiotropic effects on plant growth and development. A large number of studies have shown that the appropriate amount of melatonin supplementation is beneficial to plant growth and development by promoting root development, delaying leaf senescence, and improving fruit yield. However, the study of MT function combined with a detailed physiological analysis of nitrogen (N) absorption and metabolism in cucumber plants needs further strengthening. We performed hydroponic tests at different nitrogen levels to determine the metabolic processes associated with the enhanced tolerance to nitrogen in melatonin-treated cucumber (Cucucumis sativus L.) seedlings. Cucumber seedlings were sprayed with 100 μM melatonin or water and treated with different nitrogen in the growth chamber for 7 days. Nitrogen deficiency significantly inhibited seedling growth, and this growth inhibition was partially alleviated by melatonin. The expression analysis of related carbon and nitrogen genes showed that the genes whose expression was significantly altered by melatonin were mainly related to carbon (C) and nitrogen (N) metabolism. By enzyme activity and reactive oxygen content data analysis, melatonin-treated cucumber seedlings showed relatively stable carbon and nitrogen levels compared to untreated ones. In conclusion, MT can repair the impaired growth and development situation by regulating the nitrogen assimilation capacity and the balance between oxidation and oxidative metabolism and carbon metabolism in the cucumber under different nitrogen levels.

Kluyveromyces marxianus MTCC 1389 Augments Multi-stress Tolerance After Adaptation to Ethanol Stress.

During fermentation, yeast cells undergo various stresses that inhibit cell growth and ethanol production. Therefore, the ability to tolerate multiple stresses during fermentation is one of the important characteristics for yeast cells that can be used for commercial ethanol production. In the present study, we evaluated the multi-stress tolerance of parent and ethanol adapted Kluyveromyces marxianus MTCC1389 and their relative gene expression analysis. Multi-stress tolerance was confirmed by determining its cell viability, growth, and spot assay under oxidative, osmotic, thermal, and ethanol stress. During oxidative (0.8% H2O2) and osmotic stress (2M NaCl), there was significant cell viability of 90% and 50%, respectively, by adapted strain. On the other hand, under 45°C of thermal stress, the adapted strain was 80% viable while the parent strain was 60%. In gene expression analysis, the ethanol stress responsive gene ETP1 was significantly upregulated by 3.5 folds, the osmotic stress gene SLN1 was expressed by 3 folds, and the thermal stress responsive gene MSN2 was expressed by 7 folds. This study shows adaptive evolution for ethanol stress can develop other stress tolerances by changing relative gene expression of osmotic, oxidative, and thermal stress responsive genes.