Normalized Expression Levels Research Articles

Enrichment of glioblastoma tumor microenvironment in a GZMK+ T cell population.

2065 Background: Glioblastoma (GBM) bears a survival estimate below 10% at 5 years, despite standard chemoradiation treatment. Trials evaluating immune checkpoint blockade (ICB) in gliomas have been disappointing so far, dealing with the unique immunosuppressive tumor microenvironment (TME) of the brain. Nevertheless, we have clinical evidence of ICB efficacy for brain metastases (BrM) of immunosensitive tumors. Even if both GBMs and BrM share the same organ-specific microenvironment, it is clear that disease-specific immune characteristics instruct differential response to ICB. Methods: By using advanced deep learning algorithms to combine public and in-house-generated single-cell RNA sequencing profiles, we analyzed immune infiltrate characteristics of 602949 CD45+ cells from 76 GBM, 30 BrM from different types of extracranial tumors (13 NSCLC, 12 melanoma, 2 ovarian, 1 breast, 1 colorectal and 1 renal carcinoma) and 3 normal brain samples. The analysis was restricted to newly diagnosed GBM, excluding recurrent and pre-treated cases. CD8+ cells underwent segregation into clusters based on log2 normalized expression levels of CD3E and CD8A (with expression values greater than 0). The data integration utilized DESC, a deep learning model founded on autoencoders, and subsequent examination was conducted within the Scanpy framework. Cluster analysis was executed employing the Leiden algorithm. Differentially expressed genes (DEGs) were pinpointed across clusters using the Wilcoxon test, with False Discovery Rate (FDR) correction carried out through the Benjamini-Hochberg procedure. Results: From CD45+CD3+ cells, we isolated CD8+ T cells and identified 7 informative CD8+ T cells clusters. Based on DEGs, we could appreciate T cells clusters that were also recapitulated in other extracranial malignancies. The most abundant subset was composed of GZMK+ effector T cells (Cluster 0: GZMK, GZMM, CD44, KLRG1, IL7R, GZMA, CXCR3), followed by putative tumor-specific tissue-resident memory cells (Cluster 1: GZMB, LAG3, CTLA4, TIGIT, CXCL13, ITGAE, ENTPD1, PDCD1, CD27, HLA-DRB1), stem-cell memory-like T cells (Cluster 3: IL7R, CCR7, JUNB, KLF2, TCF7) and a less represented cluster characterized by high expression of genes belonging to the HSP family (Cluster 4: HSPA1, HSPH1, HSPE1, HSPD1, IFN). Interestingly, while the majority of the identified clusters was equally represented in the 3 different tissues, the GZMK+ effector T cells cluster (Cluster 0) was found to be more represented in GBM compared to BrM and normal brain tissue. Conclusions: A GZMK+ effector T cells subpopulation, specifically enriched in GBM, has been poorly characterized in its function and spatial localization. Our data highlight the importance of studying the composition of the immune infiltrate in GBM with multi-omics approaches in order to uncover deep mechanisms of resistance to ICB and eventually provide hints on potential therapeutic targets.

Single-cell sequencing of tumour infiltrating T cells efficiently identifies tumour-specific T cell receptors based on the T cell activation score

Adoptively transferred T cell receptor-engineered T cells are a promising cancer treatment strategy, and the identification of tumour-specific TCRs is essential. Previous studies reported that tumour-reactive T cells and TCRs could be isolated based on the expression of activation markers. However, since T cells with different cell states could not respond uniformly to activation but show a heterogeneous expression profile of activation and effector molecules, isolation of tumour-reactive T cells based on single activation or effector molecules could result in the absence of tumour-reactive T cells; thus, combinations of multiple activation and effector molecules could improve the efficiency of isolating tumour-specific TCRs. We enrolled two patients with lung adenocarcinoma and obtained their tumour infiltrating lymphocytes (TILs) and autologous tumour cells (ATCs). TILs were cocultured with the corresponding ATCs for 12 h and subjected to single-cell RNA sequencing. First, we identified three TCRs with the highest expression levels of IFNG and TNFRSF9 mRNA for each patient, yet only the top one or two recognized the corresponding ATCs in each patient. Next, we defined the activation score based on normalized expression levels of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF1 mRNA for each T cell and then identified three TCRs with the highest activation score for each patient. We found that all three TCRs in each patient could specifically identify corresponding ATCs. In conclusion, we established an efficient approach to isolate tumour-reactive TCRs based on combinations of multiple activation and effector molecules through single-cell RNA sequencing.

Time course evaluation of collagen type IV in Pectoralis major muscles of broiler chickens selected for different growth-rates

Collagen type IV (COL4) is one of the major components of animals’ and humans’ basement membranes of several tissues, such as skeletal muscles and vascular endothelia. Alterations in COL4 assembly and secretion are associated to muscular disorders in humans and animals among which growth-related abnormalities such as white striping and wooden breast affecting Pectoralis major muscles (PMs) in modern fast-growing (FG) chickens. Considering the high prevalence of these myopathies in FG broilers and that a worsening is observed as the bird slaughter age is increased, the present study was intended to evaluate the distribution and the expression level of COL4 protein and its coding genes in PMs of FG broilers at different stages of muscle development (i.e., 7, 14, 21, 28, 35, and 42 d of age). Medium-growing (MG) chickens have been considered as the control group in consideration of the lower selection pressure on breast muscle growth rate and hypertrophy. Briefly, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. The normalized expression levels of COL4 coding genes showed an overexpression of COL4A2 in FG than MG at d 28, as well as a significant decrease in its expression over their rearing period. Overall, results obtained through the gene expression analysis suggested that selection for the hypertrophic growth of FG broilers may have led to an altered regulation of fibroblast proliferation and COL4 synthesis. Moreover, western blot and IHC analyses suggested an altered secretion and/or degradation of COL4 protein in FG broilers, as evidenced by the fluctuating trend of 2 bands observed in FG over time. In view of the above, the present research supports the evidence about a potential aberrant synthesis and/or degradation of COL4 and corroborates the hypothesis regarding a likely involvement of COL4 in the series of events underlying the growth-related abnormalities in modern FG broilers.

Correlation of cancer testis antigen and interleukin expression with survival in small bowel neuroendocrine tumors.

e16247 Background: Patients with small bowel neuroendocrine tumors (SBNETs) frequently present with metastatic disease, and unfortunately, the range and efficacy of available therapies is limited. Immunotherapeutic checkpoint inhibitors have demonstrated benefit in other malignancies and may also play a role in SBNETs, although relatively little is known about the immune infiltrate in these tumors. Toward a goal of developing novel immunomodulatory strategies, we sought to evaluate the tumor immune microenvironment of SBNETs utilizing Nanostring transcriptional profiling. Methods: Patients with SBNETs who underwent surgical resection at MD Anderson Cancer Center from 2003 to 2016 were retrospectively analyzed. Clinicopathologic data was collected, and patients were stratified by survival. Overall survival (OS) from date of resection was assessed using the Kaplan-Meier method, and p-values were calculated using the log-rank test. Multivariate (MV) analysis was performed using the Cox proportional hazards model. Transcription expression from bulk RNA was analyzed using the Nanostring PanCancer Immune Profiling Panel. Results: Resected SBNETs from 45 patients were selected for transcriptional analysis. Unsupervised clustering of RNA expression data revealed separation into high and low expression groups in two distinct transcription panels: cancer testis antigens (CTA) and interleukins (IL). All patients in the IL-high group also were in the CTA-high group. CTA-high patients (n=13) had significantly improved overall survival compared to CTA-low patients (n=32; median OS not reached vs 1975 days, p=0.0032). MV analysis controlling for age, gender, metastatic disease and history of carcinoid syndrome confirmed CTA-high status as an independent predictor of improved OS (hazard ratio 6.6, 95% confidence interval 1.7-26.4, p=0.008). CTA-high patients had significantly elevated expression of CTAs (such as PRAME and GAGE1) and cytokines (such as IL2 and CXCR1). High normalized expression levels of these single genes were independent predictors of improved OS in MV analysis (PRAME p=0.008, GAGE1 p=0.006, IL2 p=0.008, CXCR1 p<0.0001). Conclusions: High expression of CTA and IL signatures in resected SBNETs identified patients with improved survival agnostic of stage. While CTA expression across multiple tumor subtypes have been implicated in their immunogenicity and potential for therapeutic targeting, this is the first work to identify a clinically relevant signal in SBNET. The concurrent increase of key cytokines (which are known to mediate anti-tumor activity) among CTA-high patients suggests an immune-mediated component to their improved survival. Further work is underway to elucidate the epigenetic mechanisms of CTA expression and silencing, with the goal of validating key CTAs such as PRAME and GAGE1 as predictive and therapeutic targets for immunologic intervention.

Effect of crude oil pollution on heavy metal content and GST gene expression of Mucuna pruriens

The study investigated the effect of crude oil pollution on the heavy metal content and gene expression of Mucuna pruriens. Varying amounts of crude oil (0ml, 200ml, 400ml, 800ml and 1000ml) were used to pollute 10kg bags of loam soil, onto which Mucuna pruriens seeds were planted for 8 weeks. Data were collected for heavy metal content of plant tissues and GST gene expression. The highest heavy metal content of the plant tissue was observed at the 800ml treatment (0.0135 g kg-1, 0.074 g kg-1, and 0.0211 g kg-1) and the least was observed for the control treatment (0.0049 g kg-1, 0.034 g kg-1 and 0.0142 g kg-1), thus showing percentage increments of 175.51%, 117.65%, and 48.59% for the copper, zinc, and nickel content respectively. This showed that the increment of crude oil pollution in the soil led to a seemingly dose-dependent increment of heavy metal content of the plant tissue. The normalized expression levels of the target gene (GST) in the calibrator (control) sample “A”, and that of the treated/polluted (test) sample “B” were established as 0.150726 and 0.145592 respectively; and, it was established that there was no significant change in the normalized expression levels of the target gene (GST) in both the control (calibrator) sample and the treated/polluted (test) sample. Crude oil treatment of M. pruriens, despite showing reductions in the morphological parameters and increments in the heavy metal content of the tissues, didn’t affect the expression of the target GST gene, as was evident in the absence of significant difference in the normalized expression levels of the target gene (GST) in both the control (calibrator) sample and the treated/polluted (test) sample. Considering the resilience of M. pruriens and its ability to adapt to the high amounts of crude oil in the soil, it is advised that it be employed in the possible phyto-extraction of crude oil and/or phyto-remediation in crude oil polluted soils.

Фактор транскрипції T-bet - діагностичний маркер розвитку некротизуючого ентероколіту в недоношених новонароджених

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that mainly affects preterm infants. The non-specificity of the clinical picture and the high percentage of complications determine the need for early diagnosis and prevention of the disease. Purpose - to establish a relationship between the T-bet expression level in the cells of the buccal mucosa, the development of HEC and the method of feeding in premature newborn infants. Materials and methods. We examined 74 newborns, who were treated in neonatal departments. We selected three groups for comparison: the Group 1 consisted of 32 newborns up to 37 weeks of gestation on breastfeeding (BF); the Group 2 - 30 preterm newborns on artificial feeding (AF); the control group - 12 full-term newborns on BF. We determined the expression level of T-bet transcription gene factor in the cells of the buccal mucosa scrapings of the newborns. Thereafter, we have calculated the T-bet normalized expression levels in the cells of the mucous membranes of infants of the Group 1 and the Group 2 in relation to the control group, where the expression level of the specified factors was taken as 1 c.u. Results. The gestational age median of the Group 1 children was 33 (31; 34) weeks; the Group 2 - 32.5 (32; 35) weeks; and it was comparatively higher versus in the Group 1 (p<0.001) - 40 (39; 41) weeks. NEC has significantly more often developed in premature children on AF than on BF (p<0.05) - 30.0±8.4% versus 9.4±5.2%. The T-bet expression in the cells of the buccal mucosa scrapings was higher in preterm children, and the highest level was in the Group 2 - 2.4 (1.98; 3.84) c.u. versus 1.48 (1.13; 2.22) c.u. in the Group 1. We have proved a direct relationship between AF and T-bet level (r=0.370; p<0.004). Pairwise comparison revealed a higher level of T-bet in children with NEC (p=0.002): 2.36 (1.94; 3.17) c.u versus 1.74 (1.01; 3.27) c.u. without signs of NEC. We found a direct correlation between the T-bet level and the development of HEC (r=0,271; p=0.021). Conclusions. The T-bet expression level in the cells of the buccal mucosa increases in newborns with the appearance of the clinical signs of NEC. The level of T-bet expression in the buccal mucosa cells can be a marker of the inflammatory process development in intestine of infants. The expression of T-bet in the buccal mucosa cells of newborns with clinical signs of NEC is higher than in full-term newborns that reflects the tendency to NEC development. We have demonstrated that the T-bet expression level is higher in preterm neonates on AF. Therefore, AF can be considered as a risk factor for the development of NEC in preterm infants. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Impaired Death Receptor Signaling Mediates Cross-Resistance to Immunotherapy in MM

The treatment landscape in various cancers is currently shifting from genotoxic drugs to immunotherapy, such as monoclonal antibodies, immunoconjugates, T-cell engaging antibodies (TCE) or CAR T-cells (CARTs). This is in particular true for multiple myeloma (MM), in which unprecedented treatment efficiencies are induced by these novel therapies; however, patients continue to relapse and a subset even faces primary resistance, with the underlying mechanisms poorly understood. The role of the death receptor signaling proteins FADD and BID in MM resistance towards immunotherapy has not yet been explored. Therefore, we first generated FADD and BID CRISPR-Cas9 knock out (KO) models in two independent MM cell lines, AMO1 and L363. We confirmed the functional impact of the KO genes on the apoptotic activity using apoptosis inducing, CD95 activating antibody (anti-FAS Ab). As hypothesized, no apoptosis was observed in our FADD KO clones following 24 hours treatment with 15µg/ml Fas antibody, compared to a 50% increase of apoptosis in the WT cells via Annexin-V PI staining. Activity of Caspase 3/7 and 8 increased 3-4 folds in the WT cells and no increase was seen in our KO models by using commercially available fluorometric assays. Detection of cleaved PARP in WT and its absence in KO clones via Western blotting further validated the inactivation of the apoptotic pathways in our KO models. Next, we treated our KO cells with 0,1µg/ml Daratumumab (Dara) along with 10:1 ratio of PBMCs from healthy donors and 5:1 and 10:1 effector to target ratio of BCMA-CARTs: Of note, killing efficacy by Daratumumab was significantly lower in our KO models compared to the WT cells (22% vs 47% cell death) after 24 hours of treatment. Similarly, BCMA-CARTs efficiency was dramatically reduced with 58% survival in our KO models compared to around 10% in WT cells. Strikingly, we did not observe such a difference in IC50 between the KO and the WT cells when we treated for 3-5 days with Bortezomib, Lenalidomide, Doxorubicin, Melphalan, or the approved ADC Belantamab mafodotin. To determine the functional profile of the CART cells we performed a commercially available cytokine quantification assay (Human IL-2, Human IFN- γ) in response to 20 hours of antigen stimulation with WT and KO cells and no difference in IL-2 and IFN-γ production was observed. In addition, we excluded CD38 or BCMA antigen loss in our KO model by Direct Stochastic Optical Reconstruction Microscopy (dSTORM) and confirmed no significant differences in receptor density /µm2 or antigen surface distribution between the WT and the KO cells. To determine the clinical relevance of our findings we screened for decreased FADD and BID expression levels in a pilot cohort of 24 newly diagnosed (NDMM) and 54 relapsed refractory MM (RRMM) patients and normalized expression levels with 26 CD138+ bone marrow plasma cell samples from healthy donors. We did not find significant expression differences between the cohorts (that did not include patients post TCE or CARTs). However, when we ranked the patients according to their gene expression of FADD and BID the ten patients with the lowest values were predominantly Daratumumab resistant (7/10 and 9/10, respectively), whereas in the patients with highest FADD BID expression Daratumumab resistance was less pronounced (3/10 and 4/10 respectively). In conclusion, our study highlights induction of apoptosis as the main mode of action of T-cell based immunotherapies in MM. Furthermore, we provide first evidence, that gene expression of FADD and BID is essential for the action of Daratumumab and BCMA CARTs but may be dispensable for the anti-tumor effects in MM of ADC, IMiD, PI or chemotherapeutic agents. In our in vivo screen, we identified patients with low expression of FADD and BID being resistant to Daratumumab, suggesting a potential resistance mechanism.

Suppressed miR-128-3p combined with TERT overexpression predicts dismal outcomes for neuroblastoma.

Molecular and clinical diversity of neuroblastomas is notorious. The activating TERT rearrangements have been associated with dismal prognosis. Suppression of miR-128-3p may complement and enhance the adverse effects of TERT overexpression. The study aimed at evaluation of prognostic significance of the miR-128-3p/TERT expression in patients with primary neuroblastoma. RNA samples isolated from fresh-frozen tumor specimens (n= 103) were reverse transcribed for evaluation of miR-128-3p and TERT expression by qPCR. The normalized expression levels were tested for correlations with the event-free survival (EFS). ROC-analysis was used to establish threshold expression levels (TLs) for the possible best prediction of the outcomes. The median follow-up was 57 months. Both TERT overexpression and miR-128-3p downregulation were independently associated with superior rates of adverse events (p= 0.027, TL =-2.32 log10 and p= 0.080, TL =-1.33 log10, respectively). The MYCN single-copy patients were stratified into groups based on the character of alterations in expression of the studied transcripts. Five-year EFS in the groups of patients with elevated TERT/normal miR-128-3p expression and normal TERT/reduced miR-128-3p expression were 0.74 ± 0.08 and 0.60 ± 0.16, respectively. The patients with elevated TERT/reduced miR-128-3p expression had the worst outcomes, with 5-year EFS of 0.40 ± 0.16 compared with 0.91 ± 0.06 for the patients with unaltered levels of both transcripts (p< 0.001). Cumulative incidence of relapse/progression for the groups constituted 0.23 ± 0.08, 0.40 ± 0.16, 0.60 ± 0.16 and 0.09 ± 0.06, respectively. Moreover, the loss of miR-128-3p was qualified as independent adverse predictor which outperformed the conventional clinical and genetic risk factors in the multivariate Cox regression model of EFS. Combined expression levels of miR-128-3p and TERT represent a novel prognostic biomarker for neuroblastoma.

Long non-coding RNA TSPEAR Antisense RNA 2 is downregulated in rheumatoid arthritis and inhibits the apoptosis of fibroblast-like synoviocytes by downregulating microRNA-212-3p (miR-212-3p)

ABSTRACT Long non-coding RNA (lncRNA) TSPEAR-AS2 (TSPEAR Antisense RNA 2) participates in many human diseases, while its roles in rheumatoid arthritis (RA) are unknown. Plasma expression levels of TSPEAR-AS2 and microRNA (miR)-212-3p in both RA patients and healthy controls were measured by RT-qPCR. Diagnostic potentials of plasma TSPEAR-AS2 and miR-212-3p were assessed by ROC curve analysis. Normalized expression levels of TSPEAR-AS2 and miR-212-3p were subjected to Pearson’s correlation coefficient to evaluate their corrections. TSPEAR-AS2 was significantly downregulated in RA patients, while plasma expression levels of miR-212-3p were significantly increased in RA patients. The expression of TSPEAR-AS2 and miR-212-3p showed promising diagnostic value for RA. Plasma expression levels of TSPEAR-AS2 and miR-212-3p were significantly and inversely correlated in RA patients but not in healthy controls. Besides, overexpression of TSPEAR-AS2 decreased the apoptosis of RA HFLSs, while miR-212-3p increased cell apoptosis. In addition, miR-212-3p attenuated the effects of overexpression of TSPEAR-AS2. Overexpression of TSPEAR-AS2 decreased the expression levels of miR-212-3p in HFLS, while overexpression of miR-212-3p did not affect the expression of TSPEAR-AS2. In conclusion, TSPEAR-AS2 is downregulated in RA and its overexpression can decrease the apoptosis of RA HFLSs by downregulating miR-212-3p.

Improved radiation expression profiling in blood by sequential application of sensitive and specific gene signatures

Purpose. Combinations of expressed genes can discriminate radiation-exposed from normal control blood samples by machine learning based signatures (with 8 to 20% misclassification rates). These signatures can quantify therapeutically-relevant as well as accidental radiation exposures. The prodromal symptoms of Acute Radiation Syndrome (ARS) overlap those present in Influenza and Dengue Fever infections. Surprisingly, these human radiation signatures misclassified gene expression profiles of virally infected samples as false positive exposures. The present study investigates these and other confounders, and then mitigates their impact on signature accuracy. Methods. This study investigated recall by previous and novel radiation signatures independently derived from multiple Gene Expression Omnibus datasets on common and rare non-malignant blood disorders and blood-borne infections (thromboembolism, S. aureus bacteremia, malaria, sickle cell disease, polycythemia vera, and aplastic anemia). Normalized expression levels of signature genes are used as input to machine learning-based classifiers to predict radiation exposure in other hematological conditions. Results. Except for aplastic anemia, these blood-borne disorders modify the normal baseline expression values of genes present in radiation signatures, leading to false-positive misclassification of radiation exposures in 8 to 54% of individuals. Shared changes, predominantly in DNA damage response and apoptosis-related gene transcripts in radiation and confounding hematological conditions, compromise the utility of these signatures for radiation assessment. These confounding conditions (sickle cell disease, thromboembolism, S. aureus bacteremia, malaria) induce neutrophil extracellular traps, initiated by chromatin decondensation, DNA damage response and fragmentation followed by programmed cell death. Riboviral infections (for example, Influenza or Dengue fever) have been proposed to bind and deplete host RNA binding proteins, inducing R-loops in chromatin. R-loops that collide with incoming replication forks can result in incompletely repaired DNA damage, inducing apoptosis and releasing mature virus. To mitigate the effects of confounders, we evaluated predicted radiation-positive samples with novel gene expression signatures derived from radiation-responsive transcripts encoding secreted blood plasma proteins whose expression levels are unperturbed by these conditions. Conclusions. This approach identifies and eliminates misclassified samples with underlying hematological or infectious conditions, leaving only samples with true radiation exposures. Diagnostic accuracy is significantly improved by selecting genes that maximize both sensitivity and specificity in the appropriate tissue using combinations of the best signatures for each of these classes of signatures.

The Na+-activated K+ channel Slack contributes to synaptic development and plasticity

Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack−/−) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6–P14) Slack−/− lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack−/−, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack−/−neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack−/−. We additionally detect a lack of mGluR-induced LTD in infant Slack−/−, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack−/−. This together with normalized expression levels of GluN2B and Rab4 hints to developmental “restoration” of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities.

Differential Expression of Key Floral Initiation Genes in Response to Plant Growth Regulator Application and Alternate Bearing in Pecan

Regarding pecan (Carya illinoinensis), alternate bearing, which is a biennial fluctuation of crop yield, is a major hindrance for the pecan industry. Little is known about the internal cues that trigger pecan shoots to become reproductive. This 2-year study approached the mysteries of alternate bearing of pecan by determining whether pecan homologs of three genes known to control floral initiation in other species are expressed differently at various times of the growing season or in distinct plant tissues, and whether expression of these genes can be manipulated by plant growth regulator (PGR) application when compared with an untreated control group. The flowering genes of interest were pecan homologs of leafy (CpLFY), apetala1 (CpAP1), and flowering locus t (CpFT). During year 1 (2014), PGRs ethephon and gibberellin GA3 were applied at the shoot level 1 week before each of three tissue sampling dates (13 June, 3 July, 29 July). During the following year (2015), two more PGRs were added to the study [a second double rate (2X) of gibberellin GA3 and ethylene inhibitor aminoethoxyvinylglycine (AVG)] for a total of four PGRs (applied on 10 June, 1 July, and 23 July) plus the untreated control. Experimental leaf and bud tissues were sampled from fruiting and nonfruiting shoots on mature ‘Western’ pecan trees and analyzed separately. Normalized expression levels of CpLFY and CpAP1 were significantly higher in buds than in leaves. Normalized expression of CpLFY in bud tissues differed statistically based on the sampling date in 2014, with the earliest date (13 June) having higher expression than the two later dates that year. In 2015, a treatment × date interaction revealed that, compared with the untreated control, CpLFY expression was significantly lower in shoots treated with both gibberellin GA3 dosages on 1 July. A few weeks later (23 July), CpLFY expression was lower in the 2X GA3 treatment group and higher in samples treated with AVG. In 2014, CpAP1 expression in buds was significant, with a treatment × date interaction in which ethephon increased CpAP1 expression, but only on one date (29 July). In 2015, bud CpAP1 expression was significantly higher in fruiting than in nonfruiting shoots; however, again, only on one date. The results reveal differential expression of these key flowering genes based on tissue type, sampling date, and fruiting status of the shoot and PGR treatment. Results suggest that more research of the effects of PGRs is necessary for understanding the flowering behavior of pecan and mitigating the intensity of alternate bearing.

Development of a method for determining the levels of normalized expression of structural and functional genes in patients with knee joint arthropathy

Objective : to develop a method of molecular genetic analysis for determining the normalized expression levels of COL2A1, COL6A1, MMP-2, and MMP-9 genes in the biological material of patients with gonarthrosis. Material and methods . The biopsy samples of knee joint cartilage of 10 patients with gonarthrosis were used as a biological material for the study. Nucleic acids were isolated from the samples of the biological material using TRIZol Reagent. Reverse transcription was performed using the SuperScript III reverse transcriptase kit, dNTP and Ribonuclease inhibitor (Invitrogen, USA). Results . The usage of VectorNTI software has made it possible to select pairs of primers (forward and reverse) and TaqMan probes for COL2A1, COL6A1, MMP-2 , and MMP-9 genes, and to study the possibility of the use of the selected pairs of primers and probes for the purpose of identification of each of the studied genes. In-house test systems in the multiplex format have been designed for reference and target genes. Conclusion . High specificity (100 %) of the selected oligonucleotide primer sets was confirmed using the online application NCBI/Blast. The developed molecular genetic method can be applied to determine the normalized expression levels of COL2A1, COL6A1, MMP-2 , and MMP-9 genes in knee joint arthropathy

Identification of a novel gene expression signature associated with overall survival in patients with lung adenocarcinoma: A comprehensive analysis based on TCGA and GEO databases

ObjectivesLung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer. Understanding the molecular mechanisms underlying tumor progression is of clinical significance. This study aimed to identify novel molecular markers associated with LUAD prognosis. Materials and MethodsRNA sequencing data from the Cancer Genome Atlas (TCGA) database of LUAD tumors and paired normal tissues, and microarray data from the Gene Expression Omnibus (GEO) database were obtained. In the TCGA dataset, differentially expressed (DE) genes were identified by comparing gene expression between early-stage tumors and normal tissue, as well as between advanced-stage and early-stage tumors. A risk score was developed using a weighted linear combination of individual dysregulated protein-coding genes that was associated with overall survival (OS). The prognostic value of the risk score was evaluated using Kaplan-Meier and multivariate Cox analysis. The gene signature was further validated using independent datasets from GEO. ResultsAmong the 68 identified DE genes, 19 were individually associated with OS in univariate analyses. A risk score was constructed for each patient based on the coefficients in multivariate Cox model and normalized expression levels of these 19 genes. LUAD patients with a low risk score had a significantly better survival than those with a high risk score (log-rank P < 0.0001). After adjusting for age, sex, clinical stage, smoking history, and treatments, the patients with a low risk score had a 81 % decreased risk for death, compared to those with a high risk score (hazard ratio 0.19, 95 % confidence interval 0.097−0.36). The significant association of the risk score with OS in LUAD patients was further validated in three independent GEO datasets. ConclusionA novel 19-gene prognostic signature based on gene expression was identified in LUAD patients. The findings further improve the understanding of LUAD prognostication and have the potential to facilitate risk-stratified disease management.

Abstract S05-03: Preserving innate memory to overcome SARS-CoV-2 infection through the mevalonate pathway

Abstract Background: Easily accessible therapies have been proposed to treat SARS-CoV-2 infection. Given that the mevalonate pathway is critical in preventing nonspecific inflammation, events that interfere in this pathway in mononuclear or epithelial lung cells might determine the outcome of infected patients. Based on its mechanism of action, we hypothesize that in patients with metastatic cancer (MC), zoledronic acid (ZA) may influence the severity of COVID-19 disease. Methods: In silico analysis compared normalized expression levels of prenylation-related genes in mononuclear and epithelial cells, between infected and noninfected SARS-CoV and SARS-CoV-2 samples, from available GEO datasets. RNA analysis was done using R v3.6.3. Counts were normalized with EdgeR, and ggpubr package and was used to compute t-tests. For analyzing samples from GSE150728, Partek Flow was used to align and normalize reads from single-cell RNA. Results: Analysis of data from the 2003 SARS outbreak (GSE1739) showed that PBMCs isolated from infected patients expressed lower levels of HMGCR, FNTA, COX10, and PGGT1B (&amp;lt;0.05). In vitro results from SARS-CoV-infected Calu-3 cells (GSE17400) showed a trend of downregulation of MVK, RABGGTA, FDFT1, FNTA, and RAB27A (n=3/group). Next, we analyzed the transcriptional response to SARS-CoV-2 in Calu-3 and A549-infected cells in vitro (GSE147507). Differential downregulation of FNTA, MVK, HMGCR, and FDPS (p&amp;lt;0.05), and only upregulation of FNTA and PGGT1B (p&amp;lt;0.05), was observed in Calu-3 cells. The same dataset offered expression data from autopsy-derived, COVID-19-infected bronchial tissue from 2 patients. Here, we found that FDFT1, FNTA, HMGCR, RABGGTA, and FDPS were downregulated &amp;gt;1.5-fold as compared to their healthy controls. Infected A549 cells showed decreased levels of MVK and RABGGTA (p&amp;lt;0.05). In SARS-CoV-2-infected A549 cells, ACE2 transduction upregulated RABGGTA, HMGCR, COX10, and RAB27B when compared to non-transduced infected cells. This suggested that downregulation of prenylation might occur upon binding of the viral spike protein to the ACE2 receptor. Finally, we analyzed single-cell RNA-seq transcripts from uninfected PBMCs (no SARS reads) isolated from acutely ill SARS-CoV-2 patients (GSE150728). Here, we found no differences in the expression of any of these genes. This suggests that disruption of the mevalonate pathway may only account to infected cells. Conclusion: In light of these findings, coronaviruses may hijack the mevalonate pathway and induce autoimmunity. Response to coronavirus infection may differ in patients with MC under treatment with ZA, statins, or other substances that interfere with prenylation-mediated small vesicle trafficking (i.e., hydroxychloroquine, nicotine). Hence, studies that investigate SARS-CoV-2 disease severity in patients under treatment with ZA are strongly needed. Citation Format: Juan Luis Gomez, Adam Brufsky. Preserving innate memory to overcome SARS-CoV-2 infection through the mevalonate pathway [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S05-03.

Genetic Roadmap for Kidney Involvement of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

The outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic with an exponential growth rate. A recent study found SARS-CoV-2 nucleocapsid protein in kidney tubules of patients infected with SARS-CoV-2 (B. Diao, C.H. Wang, R.S. Wang, Z.Q. Feng

A Comprehensive Proteomic Analysis of Metastatic Cancer Progression in a Murine Model of Tumorigenesis Using Orbitrap Tandem Mass Spectrometry

While the subject of cancer metastasis has been extensively studied, research is typically limited to a few target proteins or signaling pathways analyzed via traditional means (Western blot, ELISA, etc.). In this study we have utilized cutting edge proteomic analysis to produce a database of protein expression data across seven serially passaged mouse cell lines to explore the progression of metastasis. We hypothesized that Orbitrap tandem mass spectrometry (OTMS) would replicate association of specific proteins to metastasis and delineate previously unidentified proteins. These cell lines included the parental embryonic fibroblasts (NIH/3T3), 3T3 cells transformed with a human oncogene, T1‐A cells from a primary tumor, T2‐A cells from a slightly metastatic tumor, and T3‐PA, T3‐HA, and T4‐PA cells derived from lung and liver metastases. OTMS yielded expression data for over 3,800 proteins across all seven cell lines. These outputs were considered to reach a threshold of change if protein expression was up‐or downregulated by at least two‐fold as compared to NIH/3T3 cells. There were 50 proteins that reached threshold in all six cancerous cell lines. Of these, 12% are directly associated with cell death and 18% with differentiation. Fourteen proteins reached threshold in only the metastatic cell lines. Correlation of protein up‐ or downregulation across these lines may assist in the elucidation or delineation of signal cascades and checkpoints that result in metastasis. Indeed, some of these proteins have been identified as tumorigenic via traditional means, such as moesin, which promotes invasion and metastasis, and PIN1, which positively correlates with proliferation and anchorage independent growth. Interestingly, 152 proteins met threshold expression only in T2‐A cells while being expressed at subthreshold levels in both non‐metastatic cells and highly metastatic cells, which is over twice the number of threshold proteins that were uniquely expressed in any single cell line. As T2‐A was the first cell line isolated from a metastatic growth, these data are evidence that these proteins are necessary for the conversion of a cell from a localized tumor to a metastatic cancer cell, but which return to normalized expression levels after this initial conversion. This study has yielded an impressive proteomic database to offer insight into the development of metastasis in embryonic mouse fibroblasts. It should provide a valuable resource to researchers examining how particular proteins of interest may act within the larger context of metastatic conversion. Furthermore it validates the efficacy of OTMS proteomic analysis by identifying proteins which had previously been recognized as tumorigenic via traditional proteomic studies, while gaining an unprecedented amount of novel information on models of cancer development.Support or Funding InformationThis research was supported by a grant from Oleander Medical Technologies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Magnesium but not nicotinamide prevents vascular calcification in experimental uraemia.

Optimal phosphate control is an unmet need in chronic kidney disease (CKD). High serum phosphate increases calcification burden and is associated with mortality and cardiovascular disease in CKD. Nicotinamide (NA) alone or in combination with calcium-free phosphate binders might be a strategy to reduce phosphate levels and calcification and thus impact cardiovascular disease in CKD. We studied the effect of NA alone and in combination with magnesium carbonate (MgCO3) as a potential novel treatment strategy. CKD was induced in dilute brown non-agouti/2 mice by subtotal nephrectomy followed by a high-phosphate diet (HP) and 7 weeks of treatment with NA, MgCO3 or their combination. Control mice underwent subtotal nephrectomy and received an HP or underwent sham surgery and received standard chow plus NA. CKD mice showed increased serum fibroblast growth factor 23 and calcium-phosphate product that was normalized by all treatment regimes. NA alone increased soft tissue and vascular calcification, whereas any treatment with MgCO3 significantly reduced calcification severity in CKD. While MgCO3 supplementation alone resulted in decreased calcification severity, it resulted in increased intestinal expression of the phosphate transporters type II sodium-dependent phosphate transporter 1 (Pit-1). Combined therapy of MgCO3 and NA reduced tissue calcification and normalized expression levels of intestinal phosphate transporter proteins. In conclusion, the data indicate that NA increases while MgCO3 reduces ectopic calcification severity. Augmented expression of intestinal phosphate transporters by MgCO3 treatment was abolished by the addition of NA. However, the clinical relevance of the latter remains to be explored. Importantly, the data suggest no benefit of NA regarding treatment of calcification in addition to MgCO3.

A New in Vitro Model of GDLD by Knocking Out TACSTD2 and Its Paralogous Gene EpCAM in Human Corneal Epithelial Cells.

PurposeGelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive corneal dystrophy that causes severe vision loss. Because of its poor prognosis, there is a demand for novel treatments for GDLD. Here, we establish a new in vitro disease model of GDLD based on immortalized human corneal epithelial (HCE-T) cells.MethodsBy using transcription activator-like effector nuclease plasmids, tumor-associated calcium signal transducer 2 (TACSTD2) and its paralogous gene, epithelial cell adhesion molecule (EpCAM), were knocked out in HCE-T cells. Fluorescence-activated cell sorting was performed to obtain cells in which both TACSTD2 and EpCAM were knocked out (DKO cells). In DKO cells, the expression levels and subcellular localizations of claudin (CLDN) 1, 4, and 7, and ZO-1 were investigated, along with epithelial barrier function. By using DKO cells, the feasibility of gene therapy for GDLD was also investigated.ResultsDKO cells exhibited decreased expression and aberrant subcellular localization of CLDN1 and CLDN7 proteins, as well as decreased epithelial barrier function. Transduction of the TACSTD2 gene into DKO cells nearly normalized expression levels and subcellular localization of CLDN1 and CLDN7 proteins, while significantly increasing epithelial barrier function.ConclusionsWe established an in vitro disease model of GDLD by knocking out TACSTD2 and its paralogous gene, EpCAM, in HCE-T cells. This cell line accurately reflected pathological aspects of GDLD.Translational RelevanceWe expect that the cell line will be useful to elucidate the pathogenesis of GDLD and develop novel treatments for GDLD.

Overexpression of the Thyroid Hormone-Responsive (THRSP) Gene in the Striatum Leads to the Development of Inattentive-like Phenotype in Mice

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that affects 8–12% of children globally. Factor analyses have divided ADHD symptoms into two domains: inattention and a combination of hyperactivity and impulsivity. The identification of domain-specific genetic risk variants may help uncover potential genetic mechanisms underlying ADHD. We have previously identified that thyroid hormone-responsive (THRSP) gene expression is upregulated in spontaneously hypertensive rats (SHR/NCrl) and Wistar-Kyoto (WKY/NCrl) rats which exhibited inattention behavior. Thus, we established a line of THRSP overexpressing (OE) mice and assessed their behavior through an array of behavioral tests. The gene and protein overexpression of THRSP in the striatum (STR) was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The THRSP OE mice exhibited inattention in the novel-object recognition and Y-maze test, but not hyperactivity in the open-field test and impulsivity in the cliff-avoidance and delay-discounting task. We have also found that expression of dopamine-related genes (dopamine transporter, tyrosine hydroxylase, and dopamine D1 and D2 receptors) in the STR increased. Treatment with methylphenidate (5 mg/kg), the most commonly used medication for ADHD, improved attention and normalized expression levels of dopamine-related genes in THRSP OE mice. Our findings suggest that THRSP plays a role in the inattention phenotype of ADHD and that the THRSP OE mice may be used as an animal model to elucidate the genetic mechanisms of the disorder.