Total RNA Research Articles

PSII-3 Sheep Neutrophil Activation in Response to Garlic in Vitro

Abstract St. Croix hair sheep are known to resist parasitic infection. Parasitic resistance is in part due to the increased number and ability of neutrophil leukocytes to fight pathogens. Dietary supplements such as garlic (Allium sativum L.) are antimicrobial immune modulators that can enhance production. This study sought to evaluate the impact of fresh filtered garlic on sheep neutrophils through In vitro treatment. The study collected blood from the jugular vein of five clinically healthy female St. Croix sheep at NC A&T State University Farm and isolated neutrophils from 10 mL whole blood samples from each animal. The neutrophils were treated with fresh filtered garlic after the total cell count was measured, and 1 million cells were taken for treatment. The total protein concentration in the neutrophil supernatant was measured before and after treatment. Garlic (10 ug/mL PBS) was added to neutrophils, incubated (30 minutes, 37 °C) and centrifuged. Total RNA was extracted from the pellet using the TRIZOL RNA Isolation Protocol (Sigma-Aldrich). The RNA concentration and purity were assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc.). Pooled RNA from each group was used for cDNA synthesis, followed by qPCR analysis of the expression of 84 test genes related to the innate and adaptive immune response (RT2 profiler array) of the host. Total protein concentration in the supernatant was determined using the BCA Assay (Pierce BCA Protein Assay Kit). In vitro treatment with garlic extract resulted in increased total secreted protein concentrations from treated neutrophils, as well as upregulation of 10 genes involved in immune response and downregulation of 4 genes associated with inflammation and immune regulation. The study findings indicate that garlic stimulates gene transcription and protein secretion in neutrophils from St. Croix sheep in vitro. Studies are needed on the possible impacts on neutrophil function and animal response to infection for sustainable use of garlic in animal production.

332 Effects of Ruminal Lipopolysaccharides on Primary Bovine Ruminal Epithelial Cells

Abstract The objective of this study was to investigate the immunopotential of mixed ruminal lipopolysaccharides (LPS) on cultured primary bovine ruminal epithelial cells (RECs). Primary bovine RECs were isolated from 6 yearling steers and grown in culture for 3 experiments. In Exp. 1 the objective was to determine the immunopotential of ruminal LPS, in Exp. 2 the objective was to assess tolerance to chronic LPS exposure, and the objective of Exp. 3 was to evaluate antagonistic interactions between ruminal and E.coli LPS. In exp. 1 & 2, RECs were exposed to nonpyrogenic water (CON), 20 μg/mL of E. coli LPS (E. COLI), 10 μg/mL of mixed ruminal-LPS (RUM10), 20 μg/mL of mixed ruminal-LPS (RUM20) and 40 μg/mL of mixed ruminal-LPS (RUM40) either continuously or intermittently. For the continuous exposure, RECs underwent a 6 h exposure, while for the intermittent exposure, the procedure was 1) a 12 h continuous exposure to treatments followed by LPS removal for 24 h and then another 12 h of exposure (RPT), and 2) a 12 h continuous exposure to treatments followed by LPS removal and a recovery period of 36 h (RCV). In exp 3, RECs were exposed to nonpyrogenic water (0:0), 1 μg/mL E. coli LPS (0:1), 1 μg/mL mixed ruminal-LPS:1 μg/mL E. coli LPS (1:1), 10 μg/mL mixed ruminal-LPS :1 μg/mL E. coli LPS (10:1) and 50 μg/mL mixed ruminal-LPS :1 μg/mL E. coli LPS (50:1). Each experiment was done as a complete randomized block design with 6 REC donors. The REC donor was used as a blocking factor. Each treatment had 2 technical replicates, and treatment responses for all data were analyzed with the MIXED procedure of SAS. For all exp., total RNA was extracted from RECs and real-time quantitative PCR was performed to determine the relative expression of genes for toll-like receptors (TLR2 & TLR4), proinflammatory cytokines (TNFα, IL1β, and IL6), chemokines (CXCL2 & CXCL8), growth factor-like cytokines (CSF2 & TGFβ), and a lipid mediator (PTGS2). In exp. 1, the targeted genes were upregulated in response to E. COLI (P<0.01), while all ruminal LPS treatments resulted in a lower transcript abundance (P<0.01). Regarding RPT and RCV condition, in Exp. 2, the expression of targeted genes was downregulated or not affected in response to ruminal LPS treatments. Lastly, in Exp. 3 all targeted genes resulted in decreased transcript abundance on all ruminal LPS ratios (P<0.01). Overall, our results indicate that ruminal LPS have a limited capacity to activate the TLR4/NF-κB pathway and to induce the expression of inflammatory genes.

PSIX-20 Differential Gene Expression in Peripheral White Blood Cells of Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef heifers remains a major economic loss to the cow-calf production industry. Identifying beef heifers with superior genetic potential for improved fertility would increase profitability. Therefore, the study aimed to identify differences in transcriptome profile from peripheral white blood cells (pWBCs) of beef heifers with varying reproductive potential. To accomplish this, Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-Synch + CIDR). Fourteen days after AI, all heifers were exposed to fertile bulls for a 60-day natural breeding season. Depending on the presence or absence of conceptus 120 days post-AI, heifers were classified as fertile (pregnant heifers) or sub-fertile (non-pregnant by AI or bull-breeding). Pregnancies were terminated, and all animals in both groups were cycling when pWBCs were isolated. Total RNA was extracted from the PWBCs and subjected to the library preparation and sequencing on the Nova-Seq platform. The read counts were obtained after data filtering and quality control using FastQC v0.11.9 and MultiQC v1.12 and alignment to the Ensemble’s ARS UCD1.2 Bos taurus genome reference using STAR aligner v2.7.5 for fertile (n = 8) and sub-fertile (n = 5) beef heifers. The filtered data were subjected to differential expression analysis using DESeq2. We identified 300 significantly differentially expressed genes (DEGs) with P-value ≤ 0.05 and absolute (log2 fold change) ≥ 0.5. Among them, 34 genes including FKBP5, ADCY3, REPS1, LAMP1, CTSH, ADGRE5 were identified with padj < 0.05. DEGs were over-represented for pathways such as response to thyroid stimulus, immune response, response to lipid, neutrophil degranulation, and calcium ion binding. Some of these genes were reported as fertility-related, while the other genes are novel and have the potential to predict heifers with varying reproductive potential. A detailed understanding of the underlying biological mechanisms of the top genes and a follow-up study with a higher sample size at different time points could validate the potential candidates identified in this study.

TPO Gene Expression in Relation with Promoter SNPs in Iraqi Patients with Hyperthyroidism

Background: Thyroid peroxidase (TPO) gene mutations are one of the most common causes of thyroid disorders. Objective: To investigate the effect of genetic polymorphisms in the TPO promoter region on gene expression in early-diagnosed hyperthyroid patients. Methods: Genomic DNA was extracted from 100 blood samples (75 hyperthyroid patients and 25 healthy controls), then the TPO promoter region was amplified and sequenced for genotyping rs2071399, rs2071400, and rs2071403 SNPs. Total RNA was also isolated, and cDNA synthesis was performed to determine quantitatively the expression of TPO by using qPCR. The level of TPO antibodies in serum was determined by using an enzyme-linked immunosorbent assay (ELISA). Results: The prevalence of hyperthyroidism in women was significantly higher than in men, as were serum levels of TPO-Abs. There was a significant increase in serum TPO-Abs in hyperthyroid patients (235.29 IU/ml) compared with healthy controls. Genotypes of three SNPs (rs2071399 G/A, rs2071400 C/T, and rs2071403 A/G) in the TPO promoter region were TPO rs2071399 AG and GG, and rs2071400 CT and TT genotypes were more frequent in hyperthyroid patients. There are no significant differences between rs2071403 polymorphic and non-polymorphic genotypes among hyperthyroid patients and healthy controls. The rs2071399 G/A and rs2071400 C/T gene promoter polymorphism significantly down-regulated constitutive TPO gene expression in hyperthyroid patients, but rs2071403 A/G has no major effect on gene expression. Conclusion: There was an association between the mutation in the promoter region of TPO and the incidence of hyperthyroidism.

180 Differences in Gene Expression in Granulosa Cells Harvested from Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef replacement heifers is one of the leading contributors to economic losses in the cow-calf sector of the beef production industry. Successful reproduction is complex, involving multiple tissues and biological systems. This complexity makes identifying the leading causes of subfertility difficult. One potential source of fertility issues is poor oocyte health. As developing oocytes are dependent on granulosa cells (GCs) and physically coupled with GCs, we hypothesized that the gene expression profile would be different between the GCs from fertile heifers and those from sub-fertile heifers. The objective of this study was to identify differences in gene expression in GCs collected from beef heifers of differing fertility status, either fertile or sub-fertile. To gather this information, Angus-Simmental crossbred heifers underwent an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-synch + CIDR). At 41 days post-AI, heifers were pregnancy checked through ultrasound and palpation. Heifers pregnant to AI were classified as fertile (n = 8). Any heifers that remained non-pregnant following AI were exposed to bulls of proven fertility for 61 days. Bull-exposed heifers were evaluated for pregnancy 34 days after the bulls were removed. The non-pregnant heifers following both AI and natural service were classified as sub-fertile (n = 5). Pregnancies were terminated in fertile heifers, and heifers from both groups were allowed to resume normal cyclicity before GCs being harvested. RNA was extracted from GCs and quality was checked for each sample. Total RNA from GCs of 13 heifers was subjected to the library preparation and sequencing on the Nova-Seq platform. Through the application of DESeq2, 79 genes were identified as significantly differentially expressed genes (DEGs) with P-value < 0.05 and absolute (log2 fold change) > 0.5 including DNER, HOXD10, CD52, CXCL12, and HOXD11. DEGs, along with co-expressed genes, were over-represented in pathways associated with the regulation of MAP kinase activity, regulation of serine/threonine kinase activity, MHC protein binding, and plasma membrane signaling receptor complex. Through the pursuit of knowledge regarding the function of top DEGs, increased sample size, and more diverse sample collection time points, we may improve our understanding of the causation of heifer fertility issues. Improving our knowledge of heifer infertility has the potential to develop therapeutic options, and in turn, increase the efficiency and sustainability of cow-calf production systems.

PSV-19 Supplementation of a Multi-Strain Probiotic Increases the Expression of Biomarkers of Immunity in Savannah X Spanish Goat Blood

Abstract Probiotics are considered a safe, non-chemical alternative to antibiotics and have been shown to boost the host immune response of livestock. The present study investigated the effect of Gut Candy (GC), a multi-strain probiotic supplementation, on immune gene expression in Spanish X Savannah goats. Seven adult female Spanish X Savannah goats (n = 7) received a daily dose (28 g) of a GC (containing Lactobacillus acidophilus, Enterococcus faecium, Bacillus subtilis, Propioni bacterium freudenreichii, and Aspergillus oryza) for 60 days. The control group received no probiotics (n = 7). Blood (10 mL) was collected aseptically from the jugular vein on days 0 and 60. Total RNA was extracted from whole blood, reverse transcribed to cDNA, and quantitative real-time PCR (RT-PCR) was performed using specific primers for TLR2, TLR4, TNF-α, IL1B, IL10, and MPO. The fold changes (FC) in relative gene abundance were analyzed using Livak's method. β-actin and GAPDH were used as internal controls and for normalization. Fold changes (FC) were considered significant at -2 ≥ FC ≥ 2. After 60 days of GC supplementation, there was an increase in fold change in relative gene abundance in IL10 (FC = 3.66), TNF-α (FC = 11.7), IL1β (FC = 19.59), TLR4 (FC = 5.09), and MPO (FC = 2.05). GC also tended to increase the FC of TLR 2(FC = 1.86). The results from the study demonstrate that probiotic supplementation modulates the expression of immune genes in goats. Incorporating probiotics supplementation into the feed regimen of livestock could be beneficial to the animals' health and overall performance.

PSV-13 Characterization of the Relative Abundance of Duodenal Mrna Related to Genes Involved in Copper Absorption in Different Breeds of Sheep

Abstract The objective of this experiment was to characterize relative expression of mRNA related to genes involved in Cu absorption in the duodenum of different breeds of sheep. Opportunistic liver and small intestine samples were collected from three harvested market weight Lincoln and Suffolk wether lambs (3 to 4 months of age) fed a low-quality forage based diet. A 5 cm sample of the duodenum (15 cm aboral from the pyloric sphincter) was collected from each lamb at the time of slaughter. Samples were immediately excised, sliced open, and washed with a PBS solution. The mucosal lining of the intestine was scraped with a scalpel blade to collect enterocytes. The scraped enterocytes were placed into a conical tube containing RNAlater. Furthermore, a one cm cubed sample from the center of the right lobe of the liver was collected from each lamb, washed with PBS solution, and stored in a conical tube until liver Cu concentrations could be determined. All intestinal and liver samples were stored at 4°C until transported back to the laboratory. Total enterocyte RNA was extracted and purified using TRI reagent and RNeasy. Primers for antioxidant 1 (ATOX1), Cu transporting alpha-polypeptide Atpase (ATP7A), Cu transporting beta-polypeptide Atpase (ATP7B), and Cu transporter1 (CTR1), were designed for real time PCR analysis. Liver Cu concentrations were decreased (P < 0.05) in Lincoln (225.3 ± 23.1 mg Cu/kg DM) compared with Suffolk lambs (342.1 ± 31.2 mg Cu/kg DM). The relative mRNA expression of ATOX1 (P < 0.05), ATP7B (P < 0.01), and CTR1 (P < 0.04) in duodenal enterocytes were decreased in Lincoln compared with Suffolk lambs. These data indicate that Cu absorption from the duodenum may be different in these breeds of sheep. Further research examining the impact of dietary Cu dose on genes involved in Cu absorption in different breeds of sheep as well as identification and quantification of proteins involved in Cu absorption, is warranted.

PSII-6 Changes in Galectin Gene Expression and Secretion in Response to Gum Arabica Drench in Goats

Abstract Acacia senegal is part of the natural diet of goats. Gum Arabica (GA) is a water-soluble complex polysaccharide antimicrobial, anti-inflammatory prebiotic, derived from Acacia Senegal. Its mechanism of action has not been defined. Galectins are secreted sugar-binding animal proteins involved in the regulation of innate and adaptive immunity. They function in microbe recognition and as regulators of inflammation. This study evaluated the effect of GA on Galectin transcription. Clinically healthy Boer X Spanish goats (n = 20) were randomly assigned to two groups of 10. Goats in the treatment group received 10 mL of GA in water daily for six weeks, controls received sterile water. Blood was collected weekly from the jugular vein using acid citrate dextrose as an anticoagulant. Subsequently, the blood was centrifuged to isolate the cells and plasma. Total RNA isolation was isolated from the cell pellet using Trizol (Sigma-Aldrich). The RNA concentration (ng/μL) and purity was assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc). Galectin-1, -3 and -9 transcription was conducted using goat specific primers and housekeeping genes for real-time PCR. Fold changes were calculated using the Livak method. The Log2FC values for Galectin 1, 3, and 9 expressions were 12.47, 0.29, and 2.22 respectively. Thus, GA had varying effects on structurally distinct galectins. Galectin 1 gene (Lgals1) is an important modulator of the immune response and was highly induced. Changes in Galectin transcription can impact microbial recognition and immune activation for goat health. Further analyses are needed to evaluate the functional effects of GA for application as a prebiotic and immune modulator lain goat health.

PSII-27 Inflammation- and Immunity-Associated Gene Expression in the Liver and Whole Blood of Crossbred Beef Cattle with Divergent Residual Feed Intake Phenotype

Abstract We investigated the inflammation- and immunity-associated gene expression in the liver and whole blood of crossbred beef cattle with divergent residual feed intake phenotype to identify relevant immune-related biological processes underpinning feed efficiency in beef cattle. Low-RFI beef steers (n = 8; RFI = - 1.93 kg/d) and high-RFI beef steers (n = 8; RFI = + 2.01kg/d) were selected from a group of 56 growing crossbred beef steers (average BW = 261 ± 18.5 kg) fed a high-forage total mixed ration after a 49-d performance testing period. At the end of the 49-d testing period, liver biopsies and blood samples were collected for total RNA extraction and cDNA synthesis. The mRNA expression of 84 genes each related to innate and adaptive immunity was analyzed using pathway-focused PCR-based arrays. The mRNA expressions of genes with fold change ≥ (-) 2.0 and FDR ≤ 0.05 in the blood and liver were determined to be differentially expressed. Out of the 84 immune genes analyzed, the expression of 8 genes in the blood and 20 genes in the liver were upregulated in low-RFI, compared with high-RFI steers. Gene ontology analysis of all the differentially expressed genes in the blood and liver revealed that pathways related to pattern recognition receptor activity, detection of lipopolysaccharide, detection of external biotic stimulus, positive regulation of phagocytosis, positive regulation of vitamin metabolic process, vascular endothelial growth factor production, positive regulation of epithelial tube formation and T-helper cell differentiation were enriched (FDR < 0.05) in Low-RFI beef steers. These results suggest that low-RFI steers have a robust immune repertoire that is capable of prompt response to inflammatory stimuli. This prompt response to inflammation in the Low-RFI group may be characterized by greater protein turnover and increased epithelial cell differentiation, whereby an enhanced quantity of invariant natural killer T-cells and viability of natural killer cells could induce a quicker and more effective response to inflammatory stimuli and consequently contribute to more efficient partitioning of nutrients for growth in these animals.

403 Awardee Talk: Characterization of Rumen Microbiome and Immune Genes Expression of Crossbred Beef Steers with Divergent Residual Feed Intake Phenotypes

Abstract We investigated whole blood and hepatic mRNA expressions of immune genes and rumen microbiome of crossbred beef steers with divergent residual feed intake (RFI) phenotype to identify relevant biological processes underpinning feed efficiency in beef cattle. Low-RFI beef steers (n = 20; RFI = - 1.83 kg/d) and high-RFI beef steers (n = 20; RFI = + 2.12 kg/d) were selected from a group of 108 growing crossbred beef steers (average BW = 282 ± 30.4 kg) fed a high-forage total mixed ration after a 70-d performance testing period. At the end of the 70-d testing period, liver biopsies and blood samples were collected for total RNA extraction and cDNA synthesis. Rumen fluid samples were also collected for analysis of the rumen microbial community. The mRNA expression of 84 genes related to innate and adaptive immunity was analyzed using pathway-focused PCR-based arrays. Differentially expressed genes were determined using P-value ≤ 0.05 and fold change (FC) ≥ 1.5 (in whole blood) or ≥ 2.0 (in liver). Gene ontology analysis of the differentially expressed genes revealed that pathways related to pattern recognition receptor activity, positive regulation of phagocytosis, positive regulation of vitamin metabolic process, vascular endothelial growth factor production, positive regulation of epithelial tube formation and T-helper cell differentiation were significantly enriched (FDR < 0.05) in low-RFI steers. In the rumen, the relative abundance of PeH15, Arthrobacter, Moryella, Weissella and Muribaculaceae was enriched in low-RFI steers, while Methanobrevibacter, Bacteroidales_BS11_gut_group, Bacteroides and Clostridium_sensu_stricto_1 were reduced. Our study found that low-RFI beef steers exhibit increased mRNA expression of genes related to immune cell functions in whole blood and liver tissues, specifically those involved in pathogen recognition and phagocytosis regulation. Additionally, these low-RFI steers showed differences in relative abundance of some microbial taxa which may partially account for their improved feed efficiency compared with high-RFI steers.

Advanced glycation end products impair bone marrow mesenchymal stem cells osteogenesis in periodontitis with diabetes via FTO-mediated N6-methyladenosine modification of sclerostin

BackgroundDiabetes mellitus (DM) and periodontitis are two prevalent diseases with mutual influence. Accumulation of advanced glycation end products (AGEs) in hyperglycemia may impair cell function and worsen periodontal conditions. N6-methyladenosine (m6A) is an important post-transcriptional modification in RNAs that regulates cell fate determinant and progression of diseases. However, whether m6A methylation participates in the process of periodontitis with diabetes is unclear. Thus, we aimed to investigate the effects of AGEs on bone marrow mesenchymal stem cells (BMSCs), elucidate the m6A modification mechanism in diabetes-associated periodontitis.MethodsPeriodontitis with diabetes were established by high-fat diet/streptozotocin injection and silk ligation. M6A modifications in alveolar bone were demonstrated by RNA immunoprecipitation sequence. BMSCs treated with AGEs, fat mass and obesity associated (FTO) protein knockdown and sclerostin (SOST) interference were evaluated by quantitative polymerase chain reaction, western blot, immunofluorescence, alkaline phosphatase and Alizarin red S staining.ResultsDiabetes damaged alveolar bone regeneration was validated in vivo. In vitro experiments showed AGEs inhibited BMSCs osteogenesis and influenced the FTO expression and m6A level in total RNA. FTO knockdown increased the m6A levels and reversed the AGE-induced inhibition of BMSCs differentiation. Mechanically, FTO regulated m6A modification on SOST transcripts, and AGEs affected the binding of FTO to SOST transcripts. FTO knockdown accelerated the degradation of SOST mRNA in presence of AGEs. Interference with SOST expression in AGE-treated BMSCs partially rescued the osteogenesis by activating Wnt Signaling.ConclusionsAGEs impaired BMSCs osteogenesis by regulating SOST in an m6A-dependent manner, presenting a promising method for bone regeneration treatment of periodontitis with diabetes.

Influence of Maternal BLV Infection on miRNA and tRF Expression in Calves.

Small non-coding RNAs, such as microRNAs (miRNA) and tRNA-derived fragments (tRF), are known to be involved in post-transcriptional gene regulation. Research has provided evidence that small RNAs may influence immune development in calves. Bovine leukosis is a disease in cattle caused by Bovine Leukemia Virus (BLV) that leads to increased susceptibility to opportunistic pathogens. No research has addressed the potential influence that a maternal BLV infection may have on gene regulation through the differential expression of miRNAs or tRFs in progeny. Blood samples from 14-day old Holstein calves born to BLV-infected dams were collected. Antibodies for BLV were assessed using ELISA and levels of BLV provirus were assessed using qPCR. Total RNA was extracted from whole blood samples for small RNA sequencing. Five miRNAs (bta-miR-1, bta-miR-206, bta-miR-133a, bta-miR-133b, and bta-miR-2450d) and five tRFs (tRF-36-8JZ8RN58X2NF79E, tRF-20-0PF05B2I, tRF-27-W4R951KHZKK, tRF-22-S3M8309NF, and tRF-26-M87SFR2W9J0) were dysregulated in calves born to BLV-infected dams. The miRNAs appear to be involved in the gene regulation of immunological responses and muscle development. The tRF subtypes and parental tRNA profiles in calves born to infected dams appear to be consistent with previous publications in adult cattle with BLV infection. These findings offer insight into how maternal BLV infection status may impact the development of offspring.

Unveiling Putative Pathway Enrichment Following Crisantaspase Treatment in AML through RNA Sequencing Analysis

Background: Tumor development is intricately influenced by metabolic reprogramming, resulting in the emergence of exploitable vulnerabilities that can be targeted. Glutamine (Gln) depletion has emerged as a promising therapeutic strategy for malignancies, including AML, that heavily rely on Gln for their survival. Erwinia-derived asparaginase, also called crisantaspase, has exhibited anti-AML activity in both preclinical and clinical models by disrupting Gln supply, albeit with the persistence of residual leukemia burden. To identify cellular pathways modulated after crisantaspase-induced Gln depletion, here we report our comprehensive results using a whole transcriptome profiling approach (RNA-Seq) after crisantaspase treatment. Methods: Whole-transcriptomic profiling using RNA-Seq was performed to profile gene expression of MOLM-14 AML cells following crisantaspase treatment. RNA was isolated from each sample and total RNA was utilized for this study. The samples were sequenced at IGS using the Illumina HiSeq sequencing platform. Raw sequencing reads generated for each sample were analyzed using the CAVERN analysis pipeline. Read quality was assessed using the FastQC toolkit to ensure good-quality reads for downstream analyses. P values were generated using a Wald test implemented in DESeq2 and then corrected for multiple hypothesis testing using the Benjamini-Hochberg correction method. Results: We utilized Rylaze, an FDA-approved Erwinia crisantaspase, to deplete exogenous Gln. MOLM-14 cells were treated with Rylaze for 16h, followed by whole-transcriptome/gene expression profiling (GEP) using RNA-seq. The transcriptomic analysis provided information on the regulation of gene expression between vehicle control and Rylaze treatment in AML, as well as on the potential association with several putative pathways including serine family amino acids biosynthetic process (GO: 0009070, p-value = 2.18E-08), regulation of programmed cell death (GO:0043067, p-value = 7.96E-06), and amino acid transmembrane transport (GO:0003333, p-value = 5.10E-05). Furthermore, Figure 1 illustrates the gene ontology terms enriched by the DEGs, offering insights into the underlying molecular mechanisms involved in the response to Rylaze-induced Gln depletion in MOLM-14 cells. For example, the upregulation of genes associated with the serine family amino acid biosynthetic process may suggest a compensatory mechanism to counteract the depletion of Gln induced by Rylaze. By employing generalized linear regression models, we observed significant changes in gene expression following Rylaze (141 genes) compared to the vehicle control. This finding is further illustrated by a heatmap generated from transcriptome data analysis (Figure 2). We identified 141 significantly differentially expressed genes (DEGs); among the Rylaze-treated samples, consisting of 114 up-regulated and 27 down-regulated genes. Specifically, we focused on five genes that exhibited significant modulation in response to the treatment with Rylaze. These genes included CHAC1, ADM2, S100P, CTH and SPOCK2. These genes might play crucial roles in the cellular response to Gln depletion and could potentially serve as biomarkers or therapeutic targets in AML treatment. Validation of these observations was performed using immunoblotting techniques and quantitative real-time polymerase chain reaction (qRT-PCR), which confirmed the significant increase in CHAC1, ADM2, S100P, and CTH, as well as the significant decrease in SPOCK2 (p<0.0001) following Rylaze-induced Gln depletion. These findings contribute to a better understanding of the drug's mode of action and the impact of Gln depletion on cellular processes in AML. Conclusion: This study represents a significant contribution towards advancing our understanding of amino acid-regulated metabolic pathways in AML, with potential implications for other Gln metabolism-dependent cancers as well.

Comparative analysis of the susceptibility of Aedes aegypti and Japanese Aedes albopictus to all dengue virus serotypes

BackgroundDengue fever, caused by the dengue virus (DENV), is the most common viral infection transmitted by Aedes mosquitoes (mainly Ae. aegypti and Ae. albopictus) worldwide. Aedes aegypti is not currently established in Japan, and Ae. albopictus is the primary vector mosquito for DENV in the country, but knowledge of its viral susceptibility is limited. Therefore, we aimed to clarify the status of DENV susceptibility by comparing the infection and dissemination dynamics of Japanese Ae. albopictus to all known DENV serotypes with those of Ae. aegypti.MethodsAfter propagation of each DENV serotype in Vero cells, the culture supernatants were mixed with defibrinated rabbit blood and adenosine triphosphate, and the mixture was artificially blood-sucked by two colonies of Ae. albopictus from Japan and one colony of Ae. aegypti from a dengue-endemic country (Vietnam). After 14 days of sucking, the mosquito body was divided into two parts (thorax/abdomen and head/wings/legs) and total RNA was extracted from each sample. DENV RNA was detected in these extracted RNA samples using a quantitative RT-PCR method specific for each DENV serotype, and infection and dissemination rates were analyzed.ResultsThe Japanese Ae. albopictus colonies were susceptible to all DENV serotypes. Its infection and dissemination rates were significantly lower than those of Ae. aegypti. However, the number of DENV RNA copies in Ae. albopictus was almost not significantly different from that in Ae. aegypti. Furthermore, Japanese Ae. albopictus differed widely in their susceptibility to each DENV serotype.ConclusionsIn Japanese Ae. albopictus, once DENV overcame the midgut infection barrier, the efficiency of subsequent propagation and dissemination of the virus in the mosquito body was comparable to that of Ae. aegypti. Based on the results of this study and previous dengue outbreak trends, Ae. albopictus is predicted to be highly compatible with DENV-1, suggesting that this serotype poses a high risk for future epidemics in Japan.

Dynamic Single-Cell RNA-Seq Reveals Mechanism of Selinexor-Resistance in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm caused by BCR-ABL chimeric oncogene. The administration of tyrosine kinase inhibitors (TKI) has been shown to significantly improve the prognosis of the CML cases. TKI-resistance may lead to progression of the disease into accelerated and blast phases. To tackle this problem, there are currently several strategies that can be employed to manage or overcome the difficulties associated with TKI-resistance. Selinexor is a small molecule inhibitor that targets the nuclear transporter Exportin 1 (XPO1). This inhibition leads to disruptions in various signaling pathways involved in tumor cell cycle regulation, DNA repair, and apoptosis. In CML, Selinexor can increase the nuclear-cytoplasmic distribution ratio of BCR/ABL, cause cell cycle arrest, inhibit the proliferation of CML cell line K562, and exhibit anti-tumor properties in mouse model. When combined with imatinib, Selinexor can effectively disrupt the nuclear-cytoplasmic transport signal of leukemia stem cells, resulting in reduced formation of clones and increased apoptosis. In this work, we were aiming at investigating the mechanism of drug-resistance of Selinexor in CML cell lines K562. We performed single cell dynamic transcriptome sequence to analysis intratumor heterogeneity of subpopulation of CML and identified key gene expression changes that occur as Selinexor-resistance. We generated Selinexor-resistant (SR) lines by culturing K562 cell lines with increasing concentrations of Selinexor. At first, we conducted an analysis of differential gene expression between the parental group and various levels of SR groups. Our findings revealed differential expression patterns in MT2A, TFPI, MTND3, and HMGCS1 in total RNA, as well as MT-TW, DNAJB1, and HSPB1 in newly synthesized RNA, within both the parental and drug-resistant groups. These results suggest that mitochondrial metabolism and heat shock proteins play a significant role in the regulation of cellular drug resistance. Furthermore, we found hallmark signaling pathways of epithelial mesenchymal transition and protein secretion upregulated in SR groups. Through the application of pseudo-time analysis, it was observed that cluster 6 persisted in both the parental and drug-resistant groups, and displayed characteristics indicative of tumor stem cells. Notably, a set of distinct genes were identified that distinguish cluster 6 from the other clusters, these genes are associated with lysosomes and ferroptosis pathways by KEGG enrichment analysis. Next, it was determined that the gene set in the newly synthesized RNA exhibits a consistent upregulation by the expression pattern clustering analysis. Within this gene set, we revealed an enrichment of the endoplasmic reticulum protein processing and ferroptosis signaling pathway. Finally, to provide additional evidence for the ferroptosis-related pathway in the SR group, we observed that the expression of ferroptosis-inhibitory molecules FTH1 and SLC7A11 increased gradually with the development of drug resistance. Conversely, the expression of ferroptosis-driving molecules HMGB1 and MTDH decreased gradually. Subsequently, in vitro experiments were conducted to validate that the combination of the ferroptosis inducer RSL3 can effectively overcome Selinexor resistance. In summary, this study identified a subgroup exhibiting tumor stem cell properties. Additionally, it demonstrated that the combination of ferroptosis inducer can improve resistance to Selinexor. Through single-cell dynamic transcriptome RNA-seq technology, this investigation elucidated the mechanism of resistance to Selinexor in the K562 cell line, highlighting the potential involvement ferroptosis in drug resistance. These findings offer novel insights for the treatment of CML.

Patients with Chronic Lymphocytic Leukemia Carrying t(14;19) Display a Distinctive Transcriptomic Profile and Adverse Outcome, Which Might be Overcome Continuous Therapy with BTK Inhibitors. an Italian Campus CLL and Eric Study

Introduction Rearrangements of the BCL3 gene due to the translocation t(14;19)(q32;q13) can be identified in up to 1% of chronic lymphocytic leukemias (CLL) with stimulated karyotype and is associated with an adverse outcome. In this study, we aimed at unraveling the clinico-molecular features of CLL harboring t(14;19), including the response to novel therapies. Methods CLL with t(14;19) were collected within databases from the Italian Campus CLL , European Research Initiative on CLL, German CLL study group and Ohio State University. Treatment-free survival (TFS), time to next treatment (TTNT), overall survival (OS) curves were compared with the Log-rank tests. For RNA sequencing (RNA-seq), RNA was extracted from 10 6 purified B-cells, processed by TruSeq Stranded Total RNA Ribo-Zero Gold, sequenced by Illumina at an average read depth of 120x10 6 per sample. Linear and circular RNAs (circRNA) were identified and quantified with CirComPara2 software. Differential expression analysis was conducted imposing a 0.01 Benjamini-Hochberg adjusted p value threshold by using the DESeq2 and limma-voom R/Bioconductor packages for gene and circRNA expression, respectively. Pathway enrichment was conducted using the gene set enrichment analysis (GSEA) method from the clusterProfiler R. Results We gathered data from 88 patients with t(14;19) CLL from 16 centers, 52% were males, the median age at diagnosis was 58±13years, 93% (68/73) had an unmutated IGHV status [U-IGHV, including 25% stereotyped subset #8], 60% carried trisomy 12 (+12), 52% a complex karyotype (CK, 52% ≥3 aberration, 19% ≥5), 39% atypical phenotype (22/57, Matutes score <3) and 15% TP53 abnormality (abn, deletion and/or mutation). To dissect the role of t(14;19) in CLL pathobiology, we analyzed the transcriptomes of 25 t(14;19) samples (comparable in terms of TFS and OS to the whole cohort), 22 control CLL without t(14;19) (11 with +12 and 11 with both normal FISH and karyotype, 10 being U-IGHV) and B cells from 9 healthy donors. Among the differentially expressed genes between t(14;19) cases and control CLL, 708 were upregulated and 1230 downregulated. In particular, among the top upregulated genes we found BCL3, CD79b (confirming their atypical phenotype), CDKN2A, U2, TP63 and LAG3 whereas among the downregulated CD274 and TIGIT. Of note, the t(14;19) cases displayed a significant enrichment (p-adjusted<0.01) of distinct Gene Ontology pathways, primarily downregulation of leukocyte chemotaxis, immune effector process and cytokine-mediated signaling. Further, we detected over 23000 circRNAs expressed from 7024 genes. Most circRNAs were backspliced from known exons (97%), whereas 1.8% and 1.2% involved intronic and intergenic regions, respectively. We found 60 circRNA upregulated and 197 downregulated circRNAs in t(14;19) cases compared to control CLL, including 28 also differentially expressed against normal B cells, such as circCDK14, not annotated and generated by antisense gene. After a median follow-up of 7.6 years, the median TFS and OS were 2 and 12.6 years, respectively. In univariate analysis, no clinic-biological variables correlated with TFS and OS. The poor outcome of t(14;19) patients was similar to CLL cases without t(14;19) but harboring TP53 abn or CK5, and even shorter than CK3 or +12 patients (historic data from the Italian Campus CLL). By analyzing the first-line therapy, 28 patients received FCR/BR, 6 venetoclax-based (VEN) therapy (5 within the CLL13 trial), 12 BTK inhibitor (BTKi, 10 ibrutinib and 2 acalabrutinib), 34 other therapies. TTNT was longer with BTKi (p<0.001, Figure 1). At 3-year 59%, 67%, 100% and 35% for patients treated with FCR/BR, VEN, BTKi and others did not need a further line of therapy. In the relapse setting, 11 patients received VEN and 29 BTKi; a trend for a longer TTNT was observed with BTKi (3-year, 16% vs 64%, p=0.09) Discussion In this international retrospective study we report that t(14;19) i) is recurrent in young CLL with sterotyped #8; ii) portends a negative prognostic impact superimposable to CK5 or TP53 cases; iii) display by a distinct gene expression profile characterized by the deregulation of immune checkpoints, offering a hint for innovative therapeutic approaches, and of circRNAs, whose pathogenetic role in CLL is still unknown and deserves further investigation. Finally, for this aggressive subset of CLL, a continuous therapy with a BTKi resulted in improved treatment outcomes

Secondary Lesions and Sensitivity to Signaling Inhibitors of Pediatric iAMP21 B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction Intrachromosomal amplification of chromosome 21 (iAMP21) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children is a high-risk subtype for which targeted drugs are lacking. In this study we aimed to determine the frequency of secondary lesions and investigated the cellular sensitivity for candidate targeted drugs. Methods We performed total RNA sequencing on 28 iAMP21 and 28 B-other (negative for sentinel fusion genes) pediatric samples to determine the frequency of secondary lesions in newly diagnosed patients. A panel of 18 patient derived xenografts (PDX) of 8 primary iAMP21 ALL samples was generated, and secondary lesions were validated by PCR, RT-PCR, and whole exome sequencing. To test sensitivity, primary or PDX cells were exposed ex vivo to a concentration range of gilteritinib (FLT3 inhibitor), trametinib (MEK1/2 inhibitor), or ruxolitinib (JAK1/2 inhibitor). Results Secondary lesions in the cytokine receptor gene FLT3 were enriched in iAMP21 compared with B-other ALL including internal tandem duplications (ITD) and other activating lesions (50.0% vs. 10.7%, p=0.003). Lesions in genes encoding cytokine receptors CRLF2 and IL7R had a similar frequency between iAMP21 and B-other cases (25% vs. 17.9%, p=0.75 and 7.1% vs. 0%, p=0.49 respectively). Inactivating lesions in SH2B3, the downstream negative regulator of JAK/STAT and FLT3 signalling, were more frequent in iAMP21 cases vs. B-other (46.4% vs. 7.1%, p=0.002), while the frequency of JAK1 and JAK2 mutations did not differ (3.6 vs. 0% and 10.7 vs. 10.7% p = 1 for both). All SH2B3, CRLF2 and JAK lesions were retained in PDX samples, whereas in contrast FLT3-ITD was retained in only 2 of 5 PDX. Gilteritinib sensitivity did not differ between iAMP21 and B-other cases (median LC50 1.24 µM, range 0.33 to 4.85 µM vs. median LC50 1.21 µM, range 1.20 to 1.3 µM, p=0.95). Grouping iAMP21 cases by FLT3 and SH2B3 status, samples with both FLT3-ITD and SH2B3 lesion had the highest sensitivity to gilteritinib (median LC50 0.39 µM, range 0.35 to 0.43 µM). Samples with only FLT3 or SH2B3 lesion did not show increased sensitivity compared to those without a lesion (p>0.5 for both). Median ruxolitinib sensitivity did not statistically differ between iAMP21 and B-other cases (median LC50 8.38 µM, range 1.00 to >10 µM, vs. median LC50 0.48, range 0.21 to >10 µM; p=1) although extreme resistance to ruxolitinib seemed more frequent in iAMP21 cases (6 out of 12 cases) and was not related to FLT3 or SH2B3 status. CRLF2-rearranged ( CRLF2r) iAMP21 cases were slightly more sensitive to ruxolitinib than those lacking CRLF2r, although this difference did not reach significance (median LC50 4.72 µM, range 1.0-4.85 vs. median LC50 >10 µM, range 2.66 to >10 µM; p=0.08). The highest sensitivity was found in the only case with both a CRLF2r and a JAK1 mutation (LC50 of 1.00 µM). A large variation was present in trametinib sensitivity amongst both iAMP21 and B-other cases (median LC50 0.11 µM, range 0.017 to >5 µM vs. median LC50 0.18, range 0.018 to >5 µM; p=0.72). More than half of iAMP21 cases were sensitive irrespective of RAS-pathway lesion status. Conclusion iAMP21 leukemias are enriched in FLT3 and in SH2B3 lesions which, when co-occurring, affect sensitivity to FLT3 inhibition by gilteritinib but do not affect JAK-inhibition by ruxolitinib. This suggests these lesions act synergistically and might bypass downstream JAK/STAT signalling. This might also explain the observed sensitivity to RAS-pathway inhibition irrespective of secondary lesions. These results suggest that further research into FLT3 and RAS signalling inhibitors might lead to better treatment options for pediatric iAMP21 BCP-ALL.

Dry blood spot samples to monitor immune-associated mRNA expression in intervention studies: Impact of Baker's yeast beta glucan.

Monitoring immunological response to physical stressors in a field setting is challenging because existing methods require a laboratory visit and traditional blood collection via venipuncture. The purpose of this study was to determine if our optimized dry blood spot (DBS) methodology yields sufficient total RNA to quantify the effect of Baker's Yeast Beta Glucan supplementation (BYBG; Wellmune; 250mg/d) on post-exercise mRNA expression. Participants had venous DBS samples collected prior to (PRE), and immediately (POST), 2 (2H), and 4 (4H) hrs after completion of a 90min run/walk trial in a hot, humid environment. Total RNA extracted from DBS was analyzed using a 574-plex Human Immunology mRNA panel (Nanostring). BYBG supplementation was associated with the increased expression of 12 mRNAs (LTB4R, PML, PRFM1, TNFRSF14, LCK, MYD88, STAT3, CCR1, TNFSF10, LILRB3, MME, and STAT6) and decreased expression of 4 mRNAs (MAP4K1, IKBKG, CD5, and IL4R) across all post-exercise time points. In addition to individually changed mRNA targets, we found eleven immune-response pathways that were significantly enriched by BYBG following exercise (TNF Family signaling, immunometabolism, oxidative stress, toll-like receptor (TLR) signaling, Treg differentiation, autophagy, chemokine signaling, complement system, Th2 differentiation, cytokine signaling, and innate immune). The present approach showed that DBS samples can be used to yield useful information about mRNA biomarkers in an intervention study. We have found that BYBG supplementation induces changes at the mRNA level that support the immune system and reduce susceptibility to opportunistic infection (i.e., upper respiratory tract infection) and facilitate improved physical recovery from exercise. Future studies may look to use DBS sampling for testing other nutritional, health, or medical interventions.

Analysis of microRNA expression profiles involved oxidative stress in a deep tissue pressure injury

Abstract Objective: To investigate the association between oxidative stress and the development of deep tissue pressure injury (DTPI) at the genetic level by analyzing microRNA (miRNA) expression profiles in DTPI tissues. Methods: A model of DTPI was established in 3 adult mice and 3 elderly mice, whereas another set of 3 adult mice and 3 elderly mice was used as controls. Wound tissues were stained with hematoxylin and eosin to observe the histologic changes, and total RNA was extracted for high-throughput sequencing. Differentially expressed oxidative stress-related miRNAs were screened, and target genes were predicted using TargetScan (v5.0) and Miranda (v3.3a). Enrichment analysis of these genes was executed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. Selected differentially expressed miRNAs were further validated by quantitative real-time–polymerase chain reaction. Results: The results showed that 128 miRNAs were associated with oxidative stress, among which 86 were downregulated, and 42 were upregulated in the DTPI-elderly group; 21 were differentially expressed in the DTPI-adult group (P < 0.05). Moreover, the miRNA associated with oxidation stress between the two groups was miR-181a-1-3p. Its target genes mainly regulated MAPK and AGE pathways. quantitative real-time–polymerase chain reaction results showed that miR-181a-1-3p and miR-21a-5p were significantly downregulated in DTPI tissues. Conclusion: By analyzing miRNA expression profiles related to oxidative stress through a high-throughput sequencing system, this study sheds light on the potential pathologic mechanisms underpinning DTPI.

İnsan Trofoblast Hücre Hattında Sialik Asit Bağlayıcı Reseptörlerin (Siglecs) Ekspresyonu

Purpose: Cell surface proteins known as Siglecs exhibit a specific affinity for sialic acid. Primarily located on the surface of immune cells, Siglecs belong to the subset of lectins called I-type lectins. Siglecs have important roles in maternal-fetal immune tolerance. We aimed to analyze the expression of Siglecs on Human Villous Trophoblasts (HVT) cells.
 Materials and methods: Total RNA was extracted from the HVT cell line, cDNA was synthesized, and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed the determine the changes in Siglec -3, -5, -6, -7, -8, -9, -10, -11, and -16 mRNA levels. In addition, Siglec levels were assessed by using immunohistochemical staining. Immunoreactivity against Siglec-6 and Siglec-9 was evaluated separately according to the intensity of brown color. 
 Results: Expression levels of Siglec genes by qRT-PCR and melting curve analyses were performed using RNA extracted from the HVT cell line. Siglec -3, -5, -6, -7, -9, -10, -11, and -16 genes were found to be expressed in the HVT cell line. Differently, Siglec-8 results were undetected after cycle 40, which was considered a negative result. The immunocytochemical examination of the HVT cell line revealed that Siglec-6 expression was moderate in the cytoplasm (Score:2). Siglec-9 expression was prominent in the cytoplasm (Score:3). 
 Conclusion: The results showed that Siglec-6 and Siglec-9 were expressed more than other Siglec proteins in the human trophoblast cells. Immunocytochemistry results also support these findings. Our study is the first to show Siglec-9 expression in human trophoblast cells.