Mini Kit Research Articles

Altered Expression of Neuroplasticity-Related Genes in Alcohol Addiction and Treatment

Alcohol use disorder’s complexity arises from genetic and environmental factors, with alcohol metabolism genes and neurotransmitter pathways being critical. This study aims to analyze synaptic plasticity gene expression changes in individuals with AUD in order to study their contribution to AUD development and to identify potential biomarkers of treatment response. RNA was extracted from whole peripheral blood (20 patients, 10 healthy controls), before and after treatment (Qiagen AllPrep RNA/DNA Mini Kit), and the gene expression of 84 genes related to neuroplasticity was studied using the RT2 Profiler for Human Synaptic Plasticity RT-PCR Array (PAHS-126ZA, Qiagen), comparing AUD patients to control and responders to non-responders. The potential prognostic/predictive biomarkers were searched using machine learning models. A total of 35 dysregulated genes were found in AUD patients. EPHB2, EGR, and AKT1 were increased, while TIMP1, NCAM1, and GRM2 were decreased. Responders showed distinct gene expression profiles at baseline. After treatment, the expression of 57 genes was normalized, while NCAM1, GRM2, and BDNF showed the most significant recovery. EGR4, INHBA, and NCAM1 emerged as potential biomarkers to predict treatment success. These results indicate that gene profiles in peripheral blood can serve as prognostic markers for the prognosis and treatment of AUD, although further validation is required.

Low Detection of High-risk Human Papilloma Virus in Individuals with Ameloblastoma in a Tertiary Hospital in Lagos, Nigeria.

Ameloblastoma is a benign but aggressive epithelial odontogenic neoplasm of unknown etiology. The role of human papilloma virus (HPV) in the etiology of oral squamous cell carcinoma has prompted the investigation of HPV as an etiologic factor in ameloblastoma. This study aimed to determine the frequency of high-risk (HR) HPV in conventional ameloblastoma and the clinical parameters associated with infection. The study was approved by the ethical review boards of the institution. DNA was extracted from fresh tissue collected 750 μL of DNA/RNA Shield (Zymo Research, United States) using Invitrogen PureLink Viral RNA/DNA Mini Kit (Invitrogen, USA). The extracted DNA was assayed for the detection of 14 HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) using Anyplex™ II HPV HR Detection kit (Cat. No. HP7E00X) (Seegene Inc., Republic of Korea) on CFX-96TM Real-Time Polymerase Chain Reaction (PCR) System (Bio-Rad). Data on gender, age of patient, site of lesion, clinicohistological types of ameloblastoma and history of smoking, alcohol consumption, and practice of oral sex were collected. Data analysis was performed using analysis program SPSS version 25 and statistical significance was set at P < 0.05. Two cases of conventional ameloblastoma were positive with HPV and none of the ameloblastic carcinoma cases were positive. The HPV 16 serotype was observed in both cases. While 5 of the cases had a history of alcohol consumption, none of these cases were positive for HPV serotype. HPV 16 positivity was detected in two cases of conventional ameloblastomas and none in ameloblastic carcinoma using real-time PCR. There was no effect of exposure to smoking, alcohol consumption, and practice of oral sex and HPV in the etiology of ameloblastoma. Data available are suggestive of a limited role of HPV in the etiology of ameloblastoma.

Evaluation of tumor mutation burden (TMB) in tumor (tDNA) and plasma cell free DNA (cfDNA) in patients (pts) with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) treated with a combination of cetuximab and nivolumab (C+N).

6053 Background: High TMB is associated with immune checkpoint inhibitor (ICI) response in many solid tumors. However, obtaining sufficient tumor specimens for testing in a routine clinical setting could be a challenge. We evaluated TMB from whole-exome sequencing (WES) of cfDNA as a surrogate for tDNA TMB in R/M HNSCC pts treated with C+N. Methods: Archived formalin fixed paraffin embedded tumors, serially collected plasma (pre-treatment, on-treatment, and time of disease progression/end of treatment), peripheral mononuclear blood cells, and clinical data were obtained from R/M HNSCC pts treated with C+N from a completed clinical trial (NCT03370276). Nucleic acids were extracted using AllPrep DNA/RNA Mini Kit (Qiagen). Libraries were constructed using KAPA HyperPrep Kit with Library Amplification (KK8504) and IDT’s duplex UMI adapters. Hybridization and capture were performed using IDT’s XGen hybridization kit. Whole exome sequencing was performed using Illumina NovaSeq S4 flowcells (paired 151bp runs). Data preprocessing including point mutation calling, filtering, CNV calling, and purity and ploidy estimation were performed using the Getz Lab CGA WES Characterization pipeline. TMB for each sample was calculated based on the number of mutations with cancer cell fraction &gt; 0.75 as determined by ABSOLUTE and normalized to the 35Mb targeted for exome capture. Results: A total of 88 tDNA, 226 cfDNA, and 30 normal DNA (nDNA) from 82 pts were sequenced. To date, sequencing data from 13 matched tDNA/cfDNA/nDNA samples and 34 matched cfDNA/nDNA samples with sufficient tumor purity were available for analyses. The median TMB was 2.4 mut/Mb (range 0.46-17.5) in tDNA and 1.8 mut/Mb (range 1.1-12.6) in cfDNA. There was a high correlation between tDNA/cfDNA pairs in TMB (r = 0.826, p = 1.5x10-16, Pearson correlation). The median TMBs in tDNA and cfDNA obtained at different time points or treatment course did not differ significantly in 22 patients with at least two cfDNA timepoints (p = 0.26 paired t-test of log-transformed TMB). There was no difference between responder (R) vs. non-responder (NR) in cfDNA TMBs (two-sample t-test p = 0.7 between 2 R and 12 NR) and tDNA TMBs (two-sample t-test p = 0.25 between 6 R and 21 NR). However, tDNA TMB was higher in R compared NR in a subset of pts who did not have prior treatment with ICI (two-sample t-test p = 0.05 between 5 R and 2 NR). Conclusions: TMB in tDNA and cfDNA is highly correlated and suggests the possible use as a surrogate marker for tTMB. High TMB in cfDNA derived from ICI-naïve patients appears to have a prognostic value. Further evaluation is warranted to determine the role of cfDNA TMB as a biomarker of ICI response.

P109 Using baseline Peripheral blood mononuclear cell (PBMC) transcriptomics of Crohn Disease patients receiving Ustekinumab treatment as a tool to assess potential response at 1 year

Abstract Background Ustekinumab, a monoclonal antibody to the p40 subunit of interleukin-12 and interleukin-23, is used to treat Crohn Disease (CD) and clinical remission after one year was observed in about 50% of patients. We aimed to identify predictors for clinical response using PBMC transcriptomics obtained from Crohn Disease patients just prior to Ustekinumab treatment initiation. Methods RNA extraction from leukocytes was performed using Qiagen AllPrep RNA/DNA Mini Kit, followed by PolyA-RNA selection, fragmentation, cDNA synthesis, adaptor ligation, TruSeq RNA sample library preparation (Illumina, San Diego, CA), and paired-end 75bp sequencing. mRNA genes with transcripts per million (TPM) values above 1 in at least 20% of the samples were used. Differential gene expression was performed using DESeq2. Results We processed blood samples from 36 CD patients (13 males, [36%], median age 35 [IQR 29-41] years). Samples were obtained at baseline just before starting Ustekinumab treatment. 22/36 (61%) were define as responders and 14/36 (39%) as non-responders after 1 year based on Physician Global Assessment (PGA). Small bowel disease was significantly more common among responders 82% versus 36%, p=0.004. Principal component analysis (PCA) using the 12773 genes that passed expression filtering showed partial separation (Fig. 1). Differential gene expression between responders (n=22) and non-responders (n=14), did not show gene expression signature that passed FDR correction, however the analyses identified 68 genes including CXCL1/2/3 that were induced in non-responders vs. responders with p&amp;lt;0.05 and fold change above 1.5. Functional annotation enrichments of these 68 genes using ToppGene indicated enrichment (Fig. 2) for cytokine activity (FDR=1.98E-05), CXCR chemokine receptor binding (FDR=2.11E-05), Interleukin-10 signaling (FDR=5.03E-07), Genes encoding secreted soluble factors (FDR=1.73E-05), Myeloid Cells, granulocytes CD11b+ (FDR=6.64E-12), and Myeloid Cells Dendritic cells (FDR=1.80E-08). Conclusion No strong differences were found in the blood transcriptomics between responders and non-responders. However, among the non-responders we noted an increased inflammatory response enriched for pathways linked with cytokine activity and chemokine receptor binding and innate granulocytes and Myeloid signature. A larger cohort is required to validate and farther explore these findings. Fig. 1: Principal component analysis (PCA) using the 12773 genes that passed expression filtering showed partial separation.  Fig. 2: Functional annotation enrichments of the 68 genes induced in PBMC. 

P921 FMT in UC is associated with a decrease in Bacteroides-2 enterotype and response with baseline RNA and microbiota signatures

Abstract Background The efficacy of faecal microbiota transplantation (FMT) in UC has been reported to be donor-, patient- and procedure-dependent (Rees et al., 2022). The RESTORE-UC trial [NCT03110289] aimed to improve the outcome of FMT in patients with active UC by donor preselection on microbiota level, a strict anaerobic preparation and repeated FMT administration. The trial was prematurely stopped for futility (Caenepeel et al., 2022). We investigated changes in resp. biopsy and faecal samples obtained host transcriptomics and microbiota profiles from baseline to primary endpoint (PE) at week 8 to understand reasons for the observed lack of efficacy. Methods Active UC patients (total Mayo score 4-10 with endoscopic sub-score ³2, n=72) were randomly allocated to receive 4 anaerobic-prepared superdonor (S) FMT or autologous (A) FMT. Primary endpoint was defined as steroid-free clinical remission (Total Mayo ≤ 2, with no sub-score &amp;gt;1). Host RNA extractions were performed from mucosal biopsies collected at week 0 (n=63) and 8 (n=54) using the Qiagen AllPrep DNA/RNA Mini kit, and sequenced using Illumina HiSeq4000. Sequencing data was further processed (read-count filtering, normalization) and further analyzed using DESeq2 package. Corresponding faecal samples (resp. n=62 and n=50) were submitted to DNA extractions using MagAttract PowerMicrobiome DNA/RNA kit on an automated extraction platform, followed by library prep and 16S rDNA-sequencing using Illumina MiSeq. The obtained sequences were subjected to the DADA2 pipeline in R and the Quantitative Microbial Composition (QMP) was quantified by flow cytometry. Results Eight responders were observed: 3 after S-FMT and 5 after A-FMT, as well as 58 non-responders considering the intention-to-treat population. Responders to FMT tended to cluster at baseline (adonis p=0.34) and PCA analysis on the top 500 mucosal genes with the highest variance (Fig.1), showed a significant host effect at week 8 between responders and non-responders (adonis p&amp;lt;0.05). Likewise, PCA analysis of the currently available QMP (Fig.2) showed a trend towards clustering by response at week 0 and 8 resp. adonis p=0.18 and p=0.11). An overall decrease in Bacteroides-2 enterotype prevalence was observed (Fig.3) over both treatment groups and independently from reaching the PE (All McNemar's p=0.077). Conclusion A trend towards clustering of responders on host mRNA level and QMP was observed at baseline as well as at the primary endpoint, showing a potential role for pre-FMT patient selection by phenotype. Moreover, the dysbiotic enterotype Bacteroides-2 seems to be decreased after FMT. However, further analyses are mandatory to identify specific predictors of response and the origin of the microbiota shift.

A Fast and Easy Method to Co-extract DNA and RNA from an Environmental Microbial Sample.

We herein propose a fast and easy DNA and RNA co-extraction method for environmental microbial samples. It combines bead beating and phenol-chloroform phase separation followed by the separation and purification of DNA and RNA using the Qiagen AllPrep DNA/RNA mini kit. With a handling time of ~3 h, our method simultaneously extracted high-quality DNA (peak size >10-15‍ ‍kb) and RNA (RNA integrity number >6) from lake bacterioplankton filtered samples. The method is also applicable to low-biomass samples (expected DNA or RNA yield <50‍ ‍ng) and eukaryotic microbial samples, providing an easy option for more versatile eco-genomic applications.

Expression heterogeneity of ABC-transporter family genes and chemosensitivity genes in gastric tumor, carcinomatosis and lymph node metastases

Introduction. Metastatic tumors (particularly gastric cancer) have been found to be characterized by heterogeneity between the primary tumor and metastases. This type of heterogeneity comes to the fore when treating primary-metastatic forms of tumor and is an important reason for the low effectiveness of their treatment. In this regard, comparative analysis of ABC-transporter gene expression and chemosensitivity genes will allow to characterize to a certain extent the resistance and sensitivity of primary tumor, carcinomatosis and metastases to therapy and provide the basis for personalized treatment approach.Aim. To evaluate expression heterogeneity of ABC-transporter genes and chemosensitivity genes in gastric tumor, carcinomatosis and lymph node metastases.Materials and methods. Overall 41 patients with disseminated gastric cancer stage IV with carcinomatosis of peritoneum were included in the investigation. All patients underwent surgery according to Roux palliative gastrectomy. After surgery patients underwent chemotherapy depending on indications. RNA was isolated using RNeasy Plus mini kit (Qiagen, Germany). The expression level of ABC transporter genes (ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2) and chemosensitivity genes (BRCA1, RRM1, ERCC1, TOP1, TOP2α, TUBβ3, TYMS, GSTP1) was assessed by reverse transcription polymerase chain reaction (RT-PCR) in primary tumor, carcinomatosis and lymph node metastases.Results. The expression levels of the genes under study were shown to vary widely. For ABC transporter genes, ABCG1 (3.1 ± 1.1; max 32.0), ABCG2 (7.9 ± 2.3; max 54.1), ABCG2 (9.6 ± 3.8; max 101.0) were the most expressed genes in gastric tumor tissue, carcinomatosis and lymph node metastasis, respectively. Hyperexpression among chemosensitivity genes at all three sites was characteristic only of TOP2α (17.2 ± 6.0; max. 161.9; 10.8 ± 4.1; max. 105.1; 35.3 ± 0.8; max. 439.6, respectively). We found that TOP2α and BRCA1 gene expression levels were higher in lymph node metastasis compared with gastric tumor tissue and carcinomatosis (at p = 0.005 and p = 0.001). Whereas ABCC1 gene expression was statistically significantly higher in carcinomatosis (p = 0.03).Conclusion. Thus, a high level of expression heterogeneity is observed in gastric cancer, which affects the expression patterns of various genes in different localizations. The expression profile can be used to determine the level of heterogeneity and approach to personalized therapy tactics.

Differential Gene Expression Profile in DNA Damage Signaling Pathways, in De Novo Acute Myeloid Leukemia Patients before Induction Chemotherapy

Introduction: Acute Myeloid Leukemia (AML) is a life-threatening myeloid malignancy with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also important for refractoriness to treatment. Aim: The aim of this study is to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and to verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live (chemo resistant) cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression and statistical analysis was performed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of 11 lymphoma patients, used as controls, and from 64 AML patients at diagnosis who subsequently received chemotherapy with the 7+3 regimen. Response to first-line chemotherapy was recorded and patients were assigned in two groups: Responders (n=53 patients) and non-Responders (n=11 patients). Total RNA was isolated (RNeasy plus Mini Kit, Qiagen), quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Greenbased real-time RT-PCR (QuantiTect® Primer Assay, Qiagen) based on the ddCt method (Housekeeping gene GADPH). All samples were obtained from patients after informed consent and treated in accordance with the Declaration of Helsinki. Statistical analysis was performed using R Statistical Software (version 4.2.1). Results: Five genes were found significantly [p < 0.05, Students t-test] up-regulated in live cells from either leukemic cell lines after treatment with both agents and had a FoldChange > 2. More specifically PPP1R15A, CDKN1A and GADD45G genes were up-regulated in both live cell lines, GADD45A in live MV4-11 cells and EXO1 in live Kasumi cells (Figure 1A). The aforementioned genes were selected and their expression levels were quantified in human bone marrow samples. CDKN1A and GADD45G were found down-regulated in Responders [p=0.032 and p=0.001 respectively; Welch t-test] while PPP1R15A was significantly up-regulated [over 8-fold change, p= 2.08E-09; Welch t-test] compared to controls. Likewise, GADD45G and PPP1R15A were also significantly down and up regulated respectively, in non-Responders [p=0.08 and p=1.51E-05, Welch t-test]. Interestingly, PPP1R15A presented a 1.7-fold overexpression trend in non-Responders compared to Responders [p=0.18, Welch t-test] (Figure 1B). Conclusion: The results of our study suggest a dysregulation of the DNA damage and repair pathways in AML. Upregulation of GADD45G is known to dramatically induce apoptosis, differentiation, and growth arrest while increasing sensitivity of AML cells to chemotherapeutic drugs (Guo, Dan et al., Blood 2021). CDKN1A is involved in p53/TP53 mediated inhibition of cellular proliferation in response to DNA damage. Both were down-regulated in AML patients and are likely to play an important role in the pathogenesis of AML. PPP1R15A participates in DNA damage response facilitating the recovery of cells from stress. The up-regulation of PPP1R15A both in live cells treated with Ara-C and Cytarabine, as well as the 1.7-fold overexpression trend in non-Responders compared to Responders, supports the notion that PPP1R15A could be associated with resistance to induction treatment, which may have significant prognostic and therapeutic implications. A greater sample of AML patients both responding and not responding to chemotherapy, would be essential to delineate the role of genes involved in DNA damage and repair pathways in resistance to induction treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune-Related Gene Profile in HIV-Infected Patients with Discordant Immune Response.

In spite of many reports on persistent low CD4 T cell counts and change in immune-related gene expression level in patients with HIV infection, there is still uncertainty about significant association between gene expression level and HIV infection in patients with and without DIR. The aim of this study was to compare the expression level of CD4, CCL5, IFN-γ, STAT1, APOBEC3G, CD45, and ICAM-1 genes in HIV-1-positive patients with and without DIR. In this study, 30 HIV-1-positive patients (15 patients with and 15 patients without DIR [control group]) were included. PBMCs of the patients were collected through density radient centrifugation with Ficoll-Hypaque. RNeasy Plus Mini kit was used to extract RNA. Relative expression levels of CD4, CCL5, IFN-γ, STAT1, APOBEC3G, CD45, and ICAM-1 genes were evaluated by real-time PCR. The data were analyzed using one-way ANOVA. CD4 T cell counts were significantly lower in DIR patients than the control group (p < 0.01). While there was no significant difference in the relative expression levels of CD4, CCL5, IFN-γ, STAT1, CD45, and ICAM-1 between patients with DIR and control group, APOBEC3G expression level was significantly higher in the patients with DIR as compare to the control group (p < 0.01). Our findings suggest a significantly higher APOBEC3G expression level in patients with DIR, suggesting the potential role of APOBEC3G in patients with immunological discordance besides its suppressing role in HIV-1 infection. Confirmation of this hypothesis requires further research.

PB1787: GADD45A IS OVER-EXPRESSED IN THE BONE MARROW OF PATIENTS WITH DE NOVO ACUTE MYELOID LEUKEMIA REFRACTORY TO INDUCTION CHEMOTHERAPY

Background: Acute myeloid leukemia (AML) is a life-threatening malignant myeloid disorder with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment. Aims: The aim of this study was to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression was evaluated and analyzed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of lymphoma patients, used as controls, and from AML patients at diagnosis who subsequently received chemotherapy with the 7 + 3 regiment. Response to first-line chemotherapy was recorded. Total RNA was isolated (RNeasy plus Mini Kit, Qiagen) and quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Green-based real-time RT-PCR (QuantiTect® Primer Assay, Qiagen). Statistics were performed using linear regression. Results: The following genes were over two-fold up-regulated in live cells from leukemic cell lines after treatment with either agent: PPP1R15A and CDKN1A in both cell lines, GADD45A and GADD45G in live MV4-11 cells and EXO1 in live Kasumi cells. These genes were selected for relative quantification; 25 control samples and 38 AML samples (28 responders and 10 non-responders to treatment) were evaluated. GADD45A in AML compared to controls was found 2-fold up-regulated in treatment responders (ns) and 9-fold up-regulated in non-responders [p=0.01]. In addition, GADD45A was overexpressed in non-responders compared to responders [p=0.02]. PPP1R15A in AML compared to controls was 12-fold up-regulated in responders [p=0.001] and 16-fold up-regulated in non-responders [p=0.001]. (Figure 1) Image:Summary/Conclusion: GADD45A controls the balance between apoptosis and autophagy via the p38 and JNK/MAPK pathways and PPP1R15A participates in growth arrest and apoptosis in response to DNA damage by attenuating the translational elongation of key transcription factors. Both genes are likely to play an important role in the pathogenesis of AML. Furthermore, GADD45A overexpression appears to be related with resistance to induction treatment, which may have significant prognostic and therapeutic implications.

Abstract P1-08-09: High mid-treatment RNA disruption in patients with HER2-negative breast cancer predicts survival benefit after neoadjuvant chemotherapy

Abstract BACKGROUND: Ribosomal RNA fragmentation (RNA disruption) measured mid-therapy in breast cancer tumors has been shown to correlate with pCR and improved disease-free survival. Data from the NeoAva clinical trial was used to evaluate RNA disruption index values (RDI) for measurement of therapy efficacy. METHODS: The randomized phase II clinical trial NeoAva investigated the effects of neoadjuvant chemotherapy (FEC) in HER2-negative breast cancer patients with or without bevacizumab. Serial biopsies were taken prior to treatment and after 12 and 25 weeks of treatment. The majority of patients in this study were diagnosed with ER positive tumors (88% of the total 12-week samples). RNA isolation from frozen tumor tissue was carried out using the AllPrep DNA/RNA Mini kit with QIACube (Qiagen), and the quality of the RNA assessed (Agilent 2100 Bioanalyzer). Treatment response was measured using both pathological complete response (pCR) and residual cancer burden (RCB) at the time of surgery, and median follow-up time was 6.8 years. Using the RNA electropherogram data (Agilent Bioanalyzer) generated in this study, RDI values were assessed in pre-therapy samples (n=109), 12-week samples (n=98) and 25-week samples (n=106). RESULTS: RDI values were measured at three timepoints, however only the RDI values assessed from 12 week samples correlated with clinical outcomes. RDI values were higher in patients that achieved a RCB class of either 0 or 1 (n=28) (median RDI=2.6) compared with those patients that had an RCB class of 3 (n=17) (median RDI=1.3) (Mann-Whitney p=0.006). Out of the samples that were analyzed by the RNA disruption assay (RDA), 18 patients achieved a pCR and had a median RDI value of 2.6 compared with a median RDI of 1.8 for those who did not achieve a pCR (n=80); this finding did not reach statistical significance. RDI values measured in samples taken prior to therapy or at 25-weeks did not correlate with either RCB class or subsequent pCR. With 93% and 94% of patients having mid-therapy RDI values greater than 1.1 for RCB class of 0/1 or pCR, respectively, this was chosen as a cut point to generate Kaplan-Meier curves of recurrence free survival. These curves illustrated statistically significant improved survival (HR =2.6; 95% CI 0.8-8.1) for patients with a RDI &amp;gt; 1.1 (p=0.05). Similarly, Kaplan-Meier curves of breast cancer specific survival also demonstrated improved survival (HR = 3.1; 95% CI 0.8-11.7) for patients with a RDI &amp;gt; 1.1 (p=0.03). Kaplan-Meier curves based on pCR/no pCR were generated from the set of patients that had 12-week RDI values assessed and did not demonstrate improved survival in this small, predominantly ER+ group. No survival benefit was found based on RDI values measured prior to therapy or at 25 weeks. Patients that received bevacizumab (n=45) had significantly higher RDI values at 12 weeks (median RDI = 2.5) than patients that did not receive the drug (median RDI = 1.4) (n=53) (Mann-Whitney test p=0.0005). This overall increase in RDI values with bevacizumab resulted in improved survival for patients at a higher RDI cutoff values of 1.6 (p=0.03)(HR = 4.4; 95% CI 0.8-25.1). CONCLUSION: Taken together, our findings suggest that increased RNA disruption measured in tumor biopsies during neoadjuvant chemotherapy correlates with a survival benefit for patients and may be a more useful indicator of therapy efficacy than pCR, particularly for patients with ER positive tumors. Citation Format: Laura B Pritzker, Mads Haugland Haugen, Hedda von der Lippe Gythfeldt, Twinkle Masilamani, Gabriel Theriault, Renee St-Onge, Lavina D'costa, Ole Christian Lingjaerde, Amadeo Parissenti, Olav Engebraaten. High mid-treatment RNA disruption in patients with HER2-negative breast cancer predicts survival benefit after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-09.

Errata: Molecular survey of infectious bronchitis virus on poultry farms in Gifu Prefecture, Japan from 2021 to 2022 by RT-PCR with an enhanced level of detection sensitivity for the S1 gene

Harunobu SAITO, Keisuke NAKAGAWA, Yuko KITAMURA, Keisuke KUWATA, Eiji TANAKA Vol. 84, No. 9 (2022), pp. 1158-1159, the primer sequence should have been as follows: Error: RNAs from the samples collected were extracted by using TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) following the instructions in the manufacturers' product manuals. RT-PCR was performed using the One-Step RT-PCR Kit (TaKaRa Bio Inc., Kusatsu, Japan) and IBV-N gene specific primers (IBV-N forward, 5'-AGCACCCTTAGCAGCAACCC-3'; IBV-N reverse, 5'-ATCTTCAGTTTVGGAGGTAA-3') [25]. Correction: RNAs from the samples collected were extracted by using TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) following the instructions in the manufacturers' product manuals. RT-PCR was performed using the One-Step RT-PCR Kit (TaKaRa Bio Inc., Kusatsu, Japan) and IBV-N gene specific primers (IBV-N forward, 5'-AGCACCCTTAGCAGCAACCC-3'; IBV-N reverse, 5'-CCCGCGTGTACCTCTCTAGT-3'), which were originally designed to locate the region within the N gene with the highest degree of conservation) [25].

Association of Inherited Genotype and Severity of Clinical Presentation in Subjects with Verified Pas III Disorder.

Background:Polyglandular autoimmune syndrome type III (PAS III) is combination two most common autoimmune disease: Diabetes mellytus type 1 (DM1) and autoimmune thyroid disease (AITD).Objectives:The aims of the study were a) to define conection between polymorphism of CTLA-4 gene, rs 231775 with PAS III; b) to establish the conection of inherited genotype with severity of clinical features; and c) to estimate the rate of risk for severe clinical presentation among subgroups in study population.Methods:This research included 50 subjects with diagnosed PAS III syndrome, wich are on treatment in clinic for Nuclear medicine and andocrinology KCUS. As methods of research has used: hystory of disesase AND clinical examination. As material is used blood sample. From blood sample DNA was isolated withn Qiamp- DNA-mini kit, with accopanied protocol.Results:In our study, 50 patients with polyglandular autoimmune syndrome type III (PAS III) were examined, and in the study population had 26 female subjects and 24 male subjects. The average age of the participants was 31.64 years, and in the subgroups: group GWT (G-wild type) the average age was 30.20 years, group GM (G-mutated) 32.40 years and group GH (G-heterozygote) 30 , 60 years. Using the Chi-square test, the association between the polymorphism rs231775 and PAS-III was demonstrated, x2 (2.100) = 18.258, where p < 0.0001. Using the Chi-square test, the association between the rs231775 polymorphism and the severity of the clinical picture, x2 (2.50) = 8.531, where p< 0.0140 was proved. The CTLA-4 rs231775 genotypes were also assessed for disease severity.Conclusion:This study suggests that CTLA-4 expression plays a key role in balancing the immune system as well as the response against one’s own tissues, and thus in the regulation of autoimmune diseases.

Analysis of Enigma Gene-Signaling Pathways in Thyroid Cancer

Fine-needle aspiration (FNA) is one of the most accurate modes of obtaining thyroid nodule biopsies, however, up to 25% of biopsies still yield indeterminate results. There is an increasing number of thyroidectomies due to indeterminate nodules by FNA alone. Therefore, more accurate and time efficient diagnostic approaches for analyzing indeterminate thyroid nodules is required. Recent studies showed that Enigma is associated with different cancer types, including thyroid cancer progression and calcification through its interaction with bone morphogenic protein-1 (BMP-1) and tyrosine kinases linked to mitogen-activated protein kinase (MAPK) signaling pathway. Our published data on Enigma protein analysis with immunohistochemistry showed promising findings to discriminate malignant versus benign nodules. We also showed a thyroid cancer stage-dependent enhancement of Enigma protein expression. In this study, we are investigating Enigma at a gene expression level by quantitative reverse transcription polymerase chain reaction (RT-qPCR), which is more time-efficient, quantitative, and requires less tissue than immunohistochemistry. We extracted mRNA/DNA/proteins from fresh malignant and benign thyroid nodules using a Qiagen AllPrep DNA/RNA/Protein Mini Kit. After verification of the quantity and purity by NanoDrop, isolated mRNA was then run through Enigma-RT-qPCR. MAPK assay was done by western blotting using MAPK-antibody. Our initial results found that Enigma-mRNA expression level was 3-fold higher in malignant compared to benign thyroid tissues. This finding supports our previous protein expression data with a relative quantitative difference in Enigma-mRNA expression level between malignant and benign thyroid nodules. MAPK expression was upregulated in thyroid cancer compared to benign nodules. We conclude that Enigma-RT-qPCR can be used effectively in FNA samples derived from thyroid nodules, which could potentially enhance the diagnostic accuracy of indeterminate nodules and decrease unnecessary thyroidectomies. Furthermore, both Enigma and MAPK were highly expressed in advanced tumor in the same tissues. Future study is needed to establish the functional interaction of Enigma-MAPK activity in thyroid cancer cells.

The role of bone marrow microRNA (miR) in erythropoietic dysfunction after severe trauma

BackgroundPrevious data has shown that severe traumatic injury is associated with bone marrow dysfunction, which manifests as persistent injury-associated anemia. This study sought to identify whether the expression of erythropoiesis-related microRNAs were altered in the bone marrow of trauma patients to determine if these microRNAs play a role in persistent injury-associated anemia. MethodsBone marrow was collected from severely injured trauma patients who underwent fracture fixation as well as patients who underwent elective hip replacement. There were 27 trauma patients and 10 controls analyzed. Total RNA and microRNA were isolated from CD34-positive cells using the RNeasy Plus Mini kit, and genome-wide microRNA expression patterns were assayed. Genes with significant expression differences were found using BRB-ArrayTools with a significance of P < .01. ResultsThere were marked differences in expression of 108 microRNAs in the trauma group when compared with hip replacement patients. Four of these microRNAs play a role in regulating erythropoiesis: microRNA-150, microRNA-223, microRNA15a, and microRNA-24. These microRNAs were all upregulated significantly, with trauma/hip replacement fold changes of 1.7, 1.8, 1.2, and 1.2 respectively, and all act to suppress or regulate erythropoiesis. ConclusionAssessment of the bone marrow microRNA profile in trauma patients compared to those undergoing elective hip replacement revealed the differential expression of microRNA-150, microRNA-223, microRNA-15a, and microRNA-24. These microRNAs all play a role in decreased erythroid progenitor cell growth and provide important insight to the erythropoietic dysfunction seen after trauma.

Apolipoprotein E Polymorphism and Oxidative Stress in Human Peripheral Blood Cells: Can Physical Activity Reactivate the Proteasome System through Epigenetic Mechanisms?

Alzheimer's disease (AD) is characterized by proteasome activity impairment, oxidative stress, and epigenetic changes, resulting in β-amyloid (Aβ) production/degradation imbalance. Apolipoprotein E (ApoE) is implicated in Aβ clearance, and particularly, the ApoE ε4 isoform predisposes to AD development. Regular physical activity is known to reduce AD progression. However, the impact of ApoE polymorphism and physical exercise on Aβ production and proteasome system activity has never been investigated in human peripheral blood cells, particularly in erythrocytes, an emerging peripheral model used to study biochemical alteration. Therefore, the influence of ApoE polymorphism on the antioxidant defences, amyloid accumulation, and proteasome activity was here evaluated in human peripheral blood cells depending on physical activity, to assess putative peripheral biomarkers for AD and candidate targets that could be modulated by lifestyle. Healthy subjects were enrolled and classified based on the ApoE polymorphism (by the restriction fragment length polymorphism technique) and physical activity level (Borg scale) and grouped into ApoE ε4/non-ε4 carriers and active/non-active subjects. The plasma antioxidant capability (AOC), the erythrocyte Aβ production/accumulation, and the nuclear factor erythroid 2-related factor 2 (Nrf2) mediated proteasome functionality were evaluated in all groups by the chromatographic and immunoenzymatic assay, respectively. Moreover, epigenetic mechanisms were investigated considering the expression of histone deacetylase 6, employing a competitive ELISA, and the modulation of two key miRNAs (miR-153-3p and miR-195-5p), through the miRNeasy Serum/Plasma Mini Kit. ApoE ε4 subjects showed a reduction in plasma AOC and an increase in the Nrf2 blocker, miR-153-3p, contributing to an enhancement of the erythrocyte concentration of Aβ. Physical exercise increased plasma AOC and reduced the amount of Aβ and its precursor, involving a reduced miR-153-3p expression and a miR-195-5p enhancement. Our data highlight the impact of the ApoE genotype on the amyloidogenic pathway and the proteasome system, suggesting the positive impact of physical exercise, also through epigenetic mechanisms.

Prevalence of influenza and viral co-pathogens with severe acute respiratory infection visiting at tertiary care hospital in Nepal

Background: Respiratory infections are one of the major public health problems in Nepal. Data on prevalence and seasonality of respiratory viruses are poorly characterized and hence empirical antimicrobial agents are used in Nepal. The objective of this study was to describe the prevalence and seasonality of influenza and viral co-pathogen in elderly patients with severe acute respiratory infection. Methods and materials: A descriptive cross sectional study was conducted at Central Diagnostic Laboratory and Research Center, Nepal during the year 2017/018. A total of 882 throat & nasopharyngeal swab were collected from patient with severe acute respiratory infection (SARI) according to WHO case definition. Total nucleic acid was extracted using Pure Link viral RNA/DNA mini kit (Invitrogen) and multiplex RT-PCR assays were performed. Results: Of the total 882 specimens; respiratory viruses were found in 556 (63.0%) specimens. Human metapneumovirus 130 (14.7%) was most frequently detected in SARI cases followed by rhinovirus 121 (13.7%), respiratory syncytial virus 37 (4.2%) and enterovirus 36 (4.0%) respectively. The rate of human metapneumovirus 60 (15.7%) co-infection was high than rhinovirus 51 (13.4%), respiratory syncytial virus 13 (3.4%) and enterovirus 12 (3.1%) infection in elderly patients. Co-infection of human metapneumovirus, respiratory syncytial virus, and enterovirus were detected with influenza. Respiratory viruses were detected year-round in Nepal. Incidence of human metapneumovirus virus infection was most commonly detected in summer (July–Aug) and winter (Jan–Feb) season. Conclusion: Respiratory viruses are significantly higher in elderly patient with severe acute respiratory infection. Our findings highlight the importance of real time diagnostic facility for clinical management and minimize the use of antimicrobial agents for respiratory infections in Nepal.

Abstract 2534: The role of human 5-HTR2A in miRNA expression in the triple negative breast cancer cell line

Abstract In the United States, African American women are twice as likely to be diagnosed with triple-negative breast cancer (TNBC) than Caucasian women. African American women experience the highest death rates when diagnosed with breast cancer (Bca). TNBC is the most aggressive subtype of BCa and difficult to treat. Studies have demonstrated the expression of serotonin receptors (5-HTRs) on BCa cells and reported their role in tumor progression. The dysregulation of miRNAs has been associated with oncogenesis. miRNAs play a critical role in biological processes such as cell proliferation and death not only through targeting cell-cycle components but also by extensively regulating multiple signaling pathways. In the context of cancer, miRNAs have been observed to function as either oncogenes or tumor suppressors in cancer development. 5-Hydroxytryptamine receptor 2A (5-HTR2A), a G coupled-protein serotonin receptor, was implicated in cancer progression. In this study, we determined the miRNA expression profile related to 5-HTR2A biological activity in a TNBC cell line derived from an African American woman. The MDA-MB-468 cell line was cultured and an MTS assay performed to identify the effective concentration of (±)-DOI hydrochloride (5-HTR2A agonist) for treatment. Following treatment with 5 µM DOI, total RNA was isolated using Qiagen's AllPrep DNA/RNA Mini Kit following modified protocol for including miRNA. Total RNA was subjected to the NanoString human v3 miRNA kit targeting 800 endogenous miRNAs using color-coded probe sets. nSolver was used for differential miRNA expression and statistical analysis. For differential miRNA analysis DOI treated cells were compared with untreated MDA-MB 468 cells. The miRNA counts for each triplicate set were averaged. nSolver Analysis Software created by NanoString was used to determine fold change and statistical significance. Quality-control measures and normalization of data were also performed using the nSolver analysis package. After identifying significant (p&amp;lt;0.05) miRNAs and selecting those with a fold change greater than three, fourteen miRNAs were differentially expressed between the DOI treated and untreated BCa cell line (MDA-MB 468). The greatest differential expression observed in has-mir-205-5p. This oncogenic miRNA has been associated with cellular differentiation, migration, and proliferation. When comparing these fourteen miRNAs to the normal breast cell line (MCF-10a), only one miRNA (hsa-miR-221-3p), was found to be significant (p&amp;lt;0.05). In DOI treated TNBC (MDA-MB 468), hsa-miR-221-3p was downregulated by 3.44 and 1.81 compared to untreated TNBC (MDA-MB 468) and normal (MCF-10a), respectively. Reportedly, hsa-miR-221-3p (an oncogenic miRNA) is associated with TNBC and acts downstream of the oncogenic RAS-RAF-MAPK pathway. This study elucidates miRNAs contributing to TNBC development and progression in correlation with 5-HTR2A activity in vitro. Specifically, two oncogenic miRNAs were identified, has-mir-205-5p and hsa-miR-221-3p. Although their involvement in breast cancer progression has been studied, has-mir-205-5p and hsa-miR-221-3p should be validated as a consequence of 5-HTR2A biological activity. These miRNAS may be used as prognostic biomarkers in breast cancer management. Citation Format: Nicole L. Retland Moreland, Abrar Aloufi, Areej Alyahyawi, Afnan Shakoori, Joseph Aubee, Karl Thompson, Muneer Abbas. The role of human 5-HTR2A in miRNA expression in the triple negative breast cancer cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2534.

Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens

BackgroundCareful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and “denoising” approaches for sequencing low biomass specimens.ResultsWe found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = − 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination.ConclusionsWe provide insight into experimental design, quality control steps and “denoising” approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results.

189 Comparative transcriptomic analysis between Total RNA and mRNA isolated from feces of neonatal dairy calves

Abstract The fecal RNA method can be used to evaluate biological adaptations of the gastrointestinal tract of dairy calves through gene expression analysis. The process of RNA isolation from fecal samples presents several challenges, including the potential enrichment of prokaryotic (bacterial), RNA which can dilute the targeted eukaryotic RNA and consequently affect the sensitivity of the fecal RNA method to low expressed genes. Therefore, our objective in this study was to determine the differential eukaryotic RNA enrichment in total RNA vs mRNA from feces of healthy neonatal dairy calves. To test this, a comparative transcriptomic profiling of genes specific for epithelial cells, including cytokeratin 8 (KRT8) and aquaporin (AQP3), as well as inflammatory-related genes (TLR4 and IL1B) was performed in fecal samples collected from 6 pre-weaned Holstein calves. The total RNA was isolated from 200 mg of feces, using a Trizol based method along with the RNeasy Plus Mini Kit (Qiagen). Then, 45 μg of fecal total RNA was used to isolate mRNA through magnetic selection using Dynabeads® Oligo (dT)25 (Invitrogen). The standard curve was composite from all samples including cDNA from total RNA and mRNA. The internal control genes used in this experiment were B2M, ACTB, GAPDH, RPS9, and PPIA. Normalized gene expression data were log-transformed prior to statistical analysis using the Proc Mixed of SAS (SAS 9.4). The expression of KRT8 was greater (P = 0.03) in fecal mRNA than in fecal total RNA. A trend (P = 0.09) was observed for greater expression of TLR4 in fecal total RNA than in fecal mRNA. The expression of AQP3 and IL1B was not different. These preliminary data further confirms that fecal RNA method has potential to be used as a tool to evaluate gastrointestinal tract health in dairy calves, but further adjustments are needed to improve accuracy and robustness.