Relative Expression Analysis Research Articles

Diagnostic advancements: Isolating Mycobacterium avium ssp. paratuberculosis and unveiling its molecular identity with nested-PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis, which is currently prevalent in many parts of Iran and produces severe economic loss. It is hence necessary to identify and isolate the animals infected with this bacterium, so this research aimed to isolate MAP from milk and fecal samples of ELISA-positive animals and determine the molecular identity of isolates. After performing ELISA on 3,700 bovine blood samples, 115 samples of milk and feces were taken from ELISA-positive cattle and were cultured on Herald's egg yolk medium with and without mycobactin-J and then the acid-fastness of positive samples was determined using Ziehl-Neelsen staining. The 16S rRNA-PCR test was performed after DNA extraction to determine the molecular identity of isolates. Primers IS6110 and IS901 were employed to ensure that the isolates were not related to members of M. tuberculosis complex and M. avium, respectively. Primer IS900 was also used to determine the molecular identity of MAP isolates. Also, expression levels of MAP-related genes (IS900, ISMAP02, F57, MAP2191, MAP4027) were evaluated via qPCR. Finally, positive samples were confirmed based on the Nested-PCR. Results showed that a total of 9 isolates were obtained from the culture of 90 ELISA-positive samples. The results revealed that all grown samples were positive for acid-fastness. The 16S rRNA-PCR test revealed the 543 bp band, which confirms the presence of Mycobacterium in the samples. The PCR test with Primer IS900 generated the 398 bp fragment in the first step and the 298 bp fragment in the second step, indicating the presence of MAP in samples. Also, relative expression analysis revealed that MAP-related genes were significantly higher in ELIZA-positive samples than in negative ones. Based on the study findings, it can be concluded that MAP-infected animals can be identified by ELISA. In addition, mycobacterium can be isolated by culturing the samples on appropriate media and then its molecular identity can be determined by using nested-PCR.

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.

RNAi‐based genetically engineered rice resistant to black‐streaked dwarf virus does not show adverse genetic effects: A multi‐omics analysis

Societal Impact StatementAdvancements in science and technology enable us to address the challenges of the times: hunger, malnutrition, climate change, and dwindling natural resources. One of the most significant innovations in food and agriculture is the advent of genetic engineering, which may help us address the aforementioned challenges. Millions of people could benefit from the application of genetically engineered crops in agriculture and food. However, it is vital that biosafety assessments of unintended effects are carried out to identify any potential risks to humans, the environment, or society.Summary The study compared the extent of siRNA, transcriptomic and metabolomic changes in the leaves of four RNAi‐based genetically engineered rice lines to evaluate whether RNAi‐based genetically engineered rice that is resistant to black‐streaked dwarf virus experiences changes not seen in the non‐genetically engineered counterpart. siRNA, transcriptome, and metabolome profiling were conducted. siRNAs with different lengths and abundance were highly enriched in the transcript of rice; however, relative expression analysis of eight potential off‐target genes revealed that there was no decrease in gene expression in the RNAi‐based genetically engineered lines, meaning that no off‐target phenomena were caused as a result of siRNA derived from invert repeat sequences of rice black‐streaked dwarf virus in this study. This study identified some differentially expressed genes and differentially accumulated metabolites in the RNAi‐based genetically engineered lines; however, they were not enriched in pathways detrimental for humans, animals and the environment. Only 30 differentially expressed genes were significantly enriched in the plant hormone signal transduction pathway via a combined analysis using two‐way orthogonal partial least squares model. These genes belonging to salicylic acid, jasmonic acid, cytokinin, abscisic acid, and brassinosteroid pathway involved in pathways related to virus resistance or infection response were significantly changed. Taken together, there was no off‐target in RNAi‐based genetically engineered rice lines and may have a positive effect on virus resistance overall in this study.

Analysis of Mucosal- Associated Invariant T Cell Levels And Their Correlation With Tumor Immune Status In Patients With Brain Tumors

Introduction: Primary brain tumors refer to a heterogeneous group of tumors arising from cells within the CNS, and can be benign or malignant. In recent years, experimental evidence suggests that, despite the presence of blood-brain barrier (BBB) and lack of discrete lymphoid tissue, there are immunocompetent cells (TILs-Tumor infiltrating lymphocytes) within the brain parenchyma. Many of these cells are T-Lymphocytes known as CD8+ Mucosal-associated invariant T (MAIT) cells, which proliferate with stimulation, and are cytotoxic to tumor cells in vitro. To evaluate the frequency of unconventional MAIT cells in the setting of brain tumors by expression analysis of their three defining markers CD3D, KLRB1 (CD161) and a TCR a-chain variant Va7.2 (TRAV1-2 gene). Methodology: The study involved collecting EDTA blood samples and fresh brain tissue biopsies from selected population followed by RNA extraction and cDNA synthesis for measurement of relative expression analysis in blood and tissues samples. Expression data was generated by semi-quantitative real time PCR. Results: The outcome of this study demonstrated higher expression level of CD3D, KLRB1 (CD161) and a TCR a-chain variant Va7.2 (TRAV1-2 gene) in the blood of patients compared to the tissue samples showed little to no transmigration of these MAIT cells in the diseased tissues. Also, the expression of genes under-investigation was observed decreasing in both blood and tissue samples with the progression of tumor. Conclusion: MAIT cells might undergo depletion in the tumor micro-environment due to chronic activation induced exhaustion of these cells.

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.

The expression of VDACs and Bcl2 family genes in pituitary adenomas: clinical correlations and postsurgical outcomes.

Pituitary adenomas (PAs) are benign tumors with high prevalence and, occasionally, aggressive course. The tumorigenesis of these lesions is not completely understood at the molecular level. BAK1 and BAX proteins play fundamental roles in apoptosis and seem to interact with VDAC proteins, whose expressions have been markedly altered in cancer, impacting their prognosis. to evaluate the gene expression of VDAC1, VDAC2, BAK1 and BAX and their association with clinical and imaging characteristics in PA. Clinical-epidemiological data were collected from 117 tumor samples from patients affected by PA. Invasiveness was assessed by the Knosp scale. Gene expression was examined by real-time PCR. Relative expression analysis was performed by 2^(-DDCt) method. The sample was mainly composed of women (69/117 - 57.2%). Tumor subtypes observed were Non-Functioning (NF) (73/117 - 62.4%), Acromegaly (24/117 - 20.5%) and Cushing's Disease (CD) (20/117 - 17.1%). Compared to normal tissue, there was a significant reduction in VDAC1 expression in the Acromegaly (p=0.029) and NF (p=0.002) groups. BAX expression was lower in all groups (p <0.001; p=0.007; P =0.005). No difference was found in VDAC2 and BAK1 expression, compared to normal pituitary. Overexpression of VDAC2 occurred in PAs with post-surgical regrowth (p=0.042). A strongly negative correlation was observed in BAX and BAK1 expression in CD. The results indicated that downregulations of VDAC1 and BAX may be related to resistance to apoptosis. In contrast, overexpression of VDAC2 in regrowing PAs suggests an antiapoptotic role for this gene. In summary, the genes evaluated might be involved in the biopathology of PAs.

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.

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.

Intramuscular vitamin A injection in newborn lambs enhances antioxidant capacity and improves meat quality.

Vitamin A (VA) and its metabolite, retinoic acid (RA) possess several biological functions. This report investigated whether neonatal intramuscular VA injection affected antioxidative activity and meat quality in longissimus dorsi (LD) muscle of lambs. Lambs were injected with 0 (control) or 7,500 IU VA palmitate into the biceps femoris muscle on day 2 after birth. At 3, 12, and 32 weeks of age, blood samples were collected in the jugular vein for serum levels of RA and muscle samples were collected in the biceps femoris for analysis of relative mRNA expression of enzyme contributors to retinoid metabolism. All animals were harvested at 32 weeks of age and muscle samples were collected to explore the role of VA on the meat quality and antioxidant capacity of lambs. Our results indicated that VA increased the redness, crude protein, and crude fat (p < 0.05), without affecting moisture, ash, and amino acid composition in LD muscle (p > 0.05). In addition, VA increased catalase (CAT) activity and decreased malondialdehyde (MDA) levels in LD muscle (p < 0.05). Meanwhile, greater levels of CAT and NRF2 mRNA and protein contents with VA treatment were observed in LD muscle (p < 0.05), partly explained by the increased level of RA (p < 0.05). Collectively, our findings indicated that VA injection at birth could improve lamb meat quality by elevating the redness, crude protein, crude fat, and antioxidative capacity in LD muscle of lambs.

ALS gene overexpression and enhanced metabolism conferring Digitaria sanguinalis resistance to nicosulfuron in China.

Crabgrass (Digitaria sanguinalis) is a common malignant weed in corn fields in China. Recently, the acetolactate synthase (ALS) inhibitor, nicosulfuron, has shown decreasing efficacy against crabgrass. In order to elucidate the molecular basis of resistance to nicosulfuron in crabgrass, we conducted bioassays, combined with gene sequence analysis, relative expression and relative copy number analysis, to characterize resistance in crabgrass populations collected from Beijing, Heilongjiang, Jilin and Anhui provinces. Whole-plant dose-response results indicated that only population collected in Heilongjiang province (HLJ) had developed low level of resistance to nicosulfuron compared with the sensitive population (SD22). No known resistant mutation of ALS gene was found in HLJ population. The real-time fluorescence quantitative PCR results showed that the ALS gene copy number did not differ significantly between the HLJ and SD22 populations. However, the ALS gene expression in the HLJ was 2.07-fold higher than that of the SD22 population at 24 h after treatment with nicosulfuron. Pretreatment with the cytochrome P450 (CYP450) inhibitor malathion, piperonyl butoxide (PBO), and the glutathione S-transferase (GST) inhibitor 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) all partially reversed HLJ resistance. Among them, the synergistic effect of PBO and nicosulfuron is the most significant. This is the first report of resistance to nicosulfuron in crabgrass through ALS gene overexpression and possible metabolic resistance.

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.

SAT651 Human Adipose-derived Extracellular Vesicles (AdEVs) Increase Proinflammatory Markers In Renal And Endothelial Cells: An In Vitro Preliminary Study

Abstract Disclosure: C.A. Carvajal: None. M.P. Hernandez: None. P. Carrion: None. A. Tapia: None. J.A. Lopez: None. A. Vecchiola: None. C.E. Fardella: None. A. Sandoval-Borquez: None. During obesity, white adipose tissue (WAT), undergoes hypertrophic and hyperplastic changes by differentiation of preadipocytes into adipocytes, which increase the expression of PPARγ, FASN, FABP4, and adiponectin genes. These adipokines play a key role in obesity progression and in cell communication. WAT also causes a chronic inflammatory state that modifies the gene expression and secretome, including releasing of adipose-derived extracellular vesicles (AdEVs) that carry a specific cargo thought to modify different signaling pathways in target cells. Aim: To evaluate the effect of AdEVs in renal and endothelial cells and its inflammatory phenotype. Methods: Human SW872 preadipocytes and differentiated adipocytes were cultured and characterized by optical microscopy and Oil-Red-O stain. EVs from both preadipocytes and adipocytes were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, electron microscopy and w-blot. AdEVs (1x10^3) were added to renal HCD and endothelial EaHy926 cells for 24 hours. Expression of adipogenic differentiation genes in preadipocytes, adipocytes and their EVs, as well as the expression of inflammatory markers (IL-6 and IL-1B) were perfomed by RT-qPCR. Results: SW872 differentiated cells showed a classical adipocyte morphology and important accumulation of lipid-droplets. Isolated AdEVs have a donut shape morphology and size (50 - 150 nm) in accordance to the MISEV2018 guidelines, including also the EVs markers CD9 and Tsg101. A higher EVs concentration released from preadipocytes compared to adipocytes was observed (5.21x10^10 vs 0.44x10^10 particles/mL, p ≤0.05). The analysis of relative gene expression (2^-ΔΔCT), showed that adipocytes increase FAPB4 and adiponectin and decrease in PPARγ and FASN gene expression, with respect to preadipocytes (p ≤0.05). EVs from preadipocytes and adipocytes present similar relative expression of adiponectin, PPARγ and FASN than their parental cells. Finally, both renal or endothelial cells treated with AdEVs express higher levels of IL-6 and IL-1B that untreated cells (p≤ 0.05). Conclusion: EVs released from SW872 adipocytes and preadipocytes have similar gene expression as their parental cells. Treatment of both renal and endothelial cells with AdEVs showed an increase in inflammatory markers, as IL-6 and IL-1B. These preliminary results reveals novel insights about the impact of AdEVs in renal collecting duct and endothelial cells, which support novel roles of AdEVs -and its cargo- in human pathophysiology associated to obesity. Acknowledgements: This study was supported by grants ANID-FONDECYT 1212006, 3200646; CONICYT-FONDEQUIP EQM150023, SOCHED &amp; CETREN UC. Presentation: Saturday, June 17, 2023

Estimating the Change in Facial Subunits During Positive and Negative Facial Expression Using Three-Dimensional Stereophotogrammetry Facial Analysis.

Background: A more refined and clinically related facial expression analysis is required for patients who wish to be perceived more emotionally positive. Objective: To measure the change in skin vector and volume in facial subunits when expressing positive expression (happiness) compared with negative expressions (sadness, fear, disgust, and anger), using three-dimensional (3D) stereophotogrammetry analysis. Methods: This study took 3D photographs of 20 volunteers' face at rest and during positive and negative expression. The directions of skin vector and volume changes in each facial subregion were recorded and calculated. Results: In the positive expression, 78.3% (95% confidence interval [CI] 66.8-89.9) of the medial midfacial subregions presented superolateral vector and volume increase, whereas volume decrease in 82.5% (95% CI 78.5-86.5) of the lip subregions could be observed. In the negative expression, the vector changes were predominantly inferomedial in 26.0% (95% CI 15.4-36.5) of the forehead and 36.8% (95% CI 33.2-40.3) of the upper eyelid subregions, whereas volume increases in 34.0% (95% CI 30.4-37.7) of the upper eyelid subregions were observed. Conclusions: This 3D stereophotogrammetry analysis presents the morphological difference between the positive and negative expression.

Novel MHC BLB2 gene polymorphism and its association with IgY concentration and Newcastle disease antibody titer in IPB-D2 chickens.

This study aimed to identify the polymorphism of the B Locus Beta 2 (BLB2) gene and its association with immunoglobulin Y (IgY) concentration and Newcastle disease (ND) antibody titer; we analyzed BLB2 gene expression in different categories of ND antibody titers in IPB-D2 chickens. The total sample used was 100 IPB-D2 chickens. Blood samples were collected at 21 weeks old for an ELISA (enzyme-linked immunoassay) test, an HI (hemagglutination inhibition) test, and genotyping. The method for BLB2 polymorphism was Sanger sequencing. Analysis of BLB2 gene expression was performed using the cecal tonsil tissue of IPB-D2 chickens. Polymorphism data were analyzed using SNPstats and DNAsp (DNA Sequence Polymorphism) software. The association of the single-nucleotide polymorphisms (SNPs) with IgY concentration and ND antibody titer was analyzed using SAS software (version 9.2). The genotype mean values were compared by means of a T test. The relative mRNA expression analysis was performed using a quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that 13 SNPs were found in exon 2 and exon 3 in the BLB2 gene. As many as 4 out of the 13 SNPs were associated with IgY concentration. As many as 9 out the 13 SNPs may have changed amino acids. The Ct value showed that the expression of the BLB2 gene in IPB-D2 chickens with high ND antibody titers is higher than IPB-D2 chickens with low ND antibody titers. In conclusion, the AA genotype of g.458 T A was associated with high IgY concentrations, and the BLB2 gene presented with a high expression in IPB-D2 chickens with high ND antibody titers.

The role and expression of pro/antiangiogenic factors and microRNAs in gestational hypertension and pre-eclampsia

ObjectivePre-eclampsia and gestational hypertension are two common hypertensive disorders of pregnancy with pre-eclampsia accounting for high foetal and maternal morbidity and mortality rate. These disorders have an unknown aetiology and their hypertensive and end-organ pathophysiology may present too late in pregnancy. This makes the identification of early detection and differentiation markers vital. MicroRNAs have strongly been associated with pregnancy and their imbalance has been associated with the angiogenic dysregulation seen in pre-eclampsia. This study assesses the expression of pro- and antiangiogenic factors and their corresponding microRNAs in the maternal circulation of patients with pre-eclampsia and gestational hypertension. Study designWe analyzed angiogenic factors expression (sEng, TGF-β, VEGF) normalized against housekeeping gene β-actin and microRNAs (miRs: 210, 29B, 126) normalized against miR U6, potentially associated with pre-eclampsia and gestational hypertension using the targeted qPCR technique. These analytes were examined from early-onset (<34 weeks) (EOPE) (n = 12), late-onset (>34 weeks) (LOPE) (n = 12) pre-eclampsia, gestational hypertension (GH) (n = 12) and two gestationally matched normotensive groups (NG1 and 2) (n = 12) each in South African women of African ancestry. Group comparisons of experimental vs. control groups were assessed using t-test analysis for significance and represented as fold change expression. ResultsThe relative expression in group comparisons showed significant (p < 0.05) fold change of VEGF, TGF-β, sEng and miR126 in the EOPE vs. NG1. The GH vs. NG1 exhibited significant changes in VEGF, TGF-β, miR126, miR210 and miR29B. The LOPE vs. NG2 showed significant relative expression in all the angiogenic factors (VEGF, TGF-β and sEng). The GH vs. NG2 showed significant expression in VEGF and miR29B. The LOPE vs. EOPE showed significant fold changes in VEGF and miR210. Finally, only the GH vs. EOPE showed significant differences in miR210 and miR29B (p < 0.05). ConclusionThis study provides better insights into angiogenic factors and microRNAs specificity to the subtypes of gestational hypertensive disorders in pregnancy. Relative expression analysis of angiogenic factors and microRNAs showed possible novel characteristics of gestational hypertension, and potential common molecular and pathological profiles with pre-eclampsia. Furthermore, we postulate that sEng and miR29B could be early detection markers for pre-eclampsia and gestational hypertension, respectively.

The Role of Dietary Supplementation of Yeast Cell Walls in Response to a Campylobacter jejuni Inoculation in Broiler Chickens.

Controlling Campylobacter jejuni during broiler production is a topic of interest from a public health standpoint, as colonized birds can contaminate poultry products during processing and sicken humans if not properly cooked or handled before consumption. The aim of this study was to evaluate dietary yeast cell wall (YCW) as a potential alternative to antibiotic growth promoters with or without a C. jejuni challenge. A total of 2240 day-old Ross 708 males were randomly assigned within 8 experimental groups with a 4 × 2 factorial design, with 4 diets (negative control [CTL-], positive control [CTL+, bacitracin, 50 g/ ton], YCW constant dose [400 g/ton], and YCW step-down dose [SD, 800, 400, and 200 g/ton in the starter, grower, and finisher periods, respectively]) and with or without a Day-16 C. jejuni oral gavage challenge at a 103-colony-forming-units (CFU)/ml dose. Body weights and feed consumption were measured on Days 0, 14, 28, and 41 to determine broiler performance. Ileum tissue samples were collected from 24 birds per treatment on Days 17 and 24 (1 and 8 days postinoculation [PI]) for relative gene expression (RGE) analysis. Cecal content samples were collected from 24 birds per treatment on Days 24, 34, and 42 for C. jejuni enumeration and prevalence calculation. A total of 80 birds per treatment were processed to determine carcass yield on Day 44, and on Day 45, 16 carcass rinsates per treatment were collected for C. jejuni enumeration and prevalence calculation. The interaction between diet and inoculation did not influence growth performance (P > 0.05). However, a diet effect was observed in the starter period where birds fed SD diet had a lower feed conversion ratio than birds fed CTL- diet (P = 0.0165). Additionally, the treatment of birds inoculated with C. jejuni fed with SD had a trend to a lower feed conversion ratio during the grower period (P = 0.0550). The RGE of interleukin 1β and interleukin 10 was similar in all treatments 1 and 8 days PI. The RGE of avian beta defensin 10 was similar in all treatments on Day 1 PI, but different on Day 8 PI (P = 0.0476). All birds inoculated with C. jejuni had similar CFU per milliliter counts in the cecal contents at Days 24, 34, and 42 (P > 0.05), and all birds inoculated with phosphate-buffered saline were negative for C. jejuni after prevalence testing. After processing 1) carcass yield was similar in all treatments (P > 0.05); 2) C. jejuni-inoculated birds fed CTL- had lower CFU per milliliter counts than birds provided CTL+ and constant-dose diets (P = 0.0383); and 3) all birds inoculated with PBS were negative for Campylobacter. Overall, under the conditions of this study, the addition of YCW during a C. jejuni challenge did not have an impact on growth performance, innate immune response, cecal colonization, carcass yield, or carcass colonization after processing.

Regulation Transcriptional of Antibiotic Resistance Genes (ARGs) in Bacteria Isolated from WWTP

The incidence of antibiotics and transcriptional regulation of ARGs in isolated bacteria from wastewater needs to be explored. By HPLC, in samples of untreated wastewater, ampicillin (49.74 ± 5.70 µg/mL), chloramphenicol (0.60 ± 0.03 µg/mL), tylosin (72.95 ± 2.03 µg/mL), and oxytetracycline (0.22 ± 0.01 µg/mL) was determined. Through metagenomic analysis identified 58 bacterial species belonging to 9 phyla and at least 14 species have shown resistance to a variety of antibiotics. Twenty-two bacterial isolates were proved to be resistant to fifteen antibiotics of new generation and used in medical research to combat infectious diseases. Fourteen strains were shown to harbor plasmids in size ranges of 2–5 Kb, 6–10 Kb and plasmids with size greater than 10 Kb. By quantitative PCR it was possible to identify genes sul, qnr, cat1, aadA1, and sat-1 gene were shown to be present in gDNA samples from treated and untreated samples of wastewater and by relative expression analysis, differential expression of cat1, ermB, act, and tetA genes was demonstrated in strains that showed identity with Escherichia coli, Bacteroides fragilis, and Salmonella thyphi, and that were stressed with different concentrations of antibiotics. The presence of ARGs in untreated water samples, as well as in bacterial isolates, was indicative that in these habitats there are microorganisms that can resist β-lactams, aminoglycosides, tetracyclines, sulfonamides, and quinolones.

LRP5, SLC6A3, and SOX10 Expression in Conventional Ameloblastoma.

The objective of this study was the identification of key genes and inhibitory drugs related to the cell proliferation and invasion of ameloblastoma using bioinformatic analysis. The H10KA_07_38 gene profile database was analyzed by Rstudio and ShinyGO Gene Ontology enrichment. String, Cytoscape-MCODE, and Kaplan-Meier plots were generated, which were subsequently validated by RT-qPCR relative expression and immunoexpression analyses. To propose specific inhibitory drugs, a bioinformatic search using Drug Gene Budger and DrugBank was performed. A total of 204 significantly upregulated genes were identified. Gene ontology enrichment analysis identified four pathways related to cell proliferation and cell invasion. A total of 37 genes were involved in these pathways, and 11 genes showed an MCODE score of ≥0.4; however, only SLC6A3, SOX10, and LRP5 were negatively associated with overall survival (HR = 1.49 (p = 0.0072), HR = 1.55 (p = 0.0018), and HR = 1.38 (p = 0.025), respectively). The RT-qPCR results confirmed the significant differences in expression, with overexpression of >2 for SLC6A3 and SOX10. The immunoexpression analysis indicated positive LRP5 and SLC6A3 expression. The inhibitory drugs bioinformatically obtained for the above three genes were parthenolide and vorinostat. We identify LRP5, SLC6A3, and SOX10 as potentially important genes related to cell proliferation and invasion in the pathogenesis of ameloblastomas, along with both parthenolide and vorinostat as inhibitory drugs that could be further investigated for the development of novel therapeutic approaches against ameloblastoma.

Etiology behind canine uterine inertia: Role of uterine expression of MLCK4, MYH2, and PKC genes

Dystocia is an obstetrical emergency, and primary uterine inertia (PUI) is the major etiological reason among the more prevalent maternal causes in dogs. The present study involved the relative expression analysis of genes associated with myometrial contraction in medium-sized dog breeds with uterine inertia. Dogs without any progress in the parturition process even after four hours of the onset of labor and the absence of uterine contractions were considered to have complete primary uterine inertia (CPUI, n = 9). Dogs that had expelled at least one fetus and made no further progress in parturition in the absence of active uterine contraction were considered to be experiencing partial primary uterine inertia (PPUI, n = 6). Dogs with the fetal cause of dystocia (FCD), i.e., obstructive dystocia, were taken as the third (n = 7) group. Uterine tissue samples were collected during cesarean section in each group, RNA was isolated, and the relative expression of myometrial ACTA2, ACTG2, MLCK4, MYH2, and PKC genes was analyzed. The MLCK4 gene expression was downregulated in CPUI (P ≤ 0.05) and PPUI (P ≤ 0.01) when compared to FCD. The MYH2 gene expression was downregulated in PPUI in comparison to CPUI (P ≤ 0.01) and FCD (P ≤ 0.05). The PKC gene expression was upregulated in PPUI in comparison to FCD and CPUI (P ≤ 0.05). The downregulation of MLCK4 and MYH2 gene expressions recorded in PPUI indicated the possibility of myometrial defects. The possibility of myometrial defects was also observed in CPUI, but to a lesser degree, suggesting other etiologies.

Functional Additives in a Selected European Sea Bass (Dicentrarchus labrax) Genotype: Effects on the Stress Response and Gill Antioxidant Response to Hydrogen Peroxide (H2O2) Treatment.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚβ2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.