mRNA Copy Number Research Articles

Analysis of Il36a induction by C/EBP\u03b2 via a half-CRE\u2022C/EBP element in murine macrophages in dependence of its CpG methylation level

Interleukin-36α is a novel member of the IL-1 cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types after induction. The transcription factor (TF) C/EBPβ binds specifically to an essential half-CRE•C/EBP motif in the Il36a promoter to induce Il36a expression upon LPS stimulation. C/EBPs regulate gene expression by binding to recognition sequences that can contain 5′-cytosine-phosphate-guanine-3′ dinucleotides (CpG), whose methylation can influence TF binding and gene expression. Herein we show that the half-CRE•C/EBP element in the Il36a promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. We demonstrate that C/EBPβ binding to the half-CRE•C/EBP element in the Il36a promoter following LPS stimulation is insensitive to CpG methylation and that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36a promoter activity which correlated with similar Il36a mRNA copy numbers and pro-IL-36α protein amount in both cell types. Taken together, our data indicate that C/EBPβ binding to the half-CRE•C/EBP element and subsequent gene activation occurs independently of the CpG methylation status of the half-CRE•C/EBP motif and underlines the potential of C/EBPs to recognize methylated as well as unmethylated motifs.

Detection of mRNA Expression and Copy Number Variations Within the Goat Fec B Gene Associated With Litter Size.

The Booroola fecundity (FecB) gene, as the first major fecundity gene identified in Booroola sheep, has attracted careful attention. So far, previous research have uncovered the FecB mutation (Q249R) as the main mutation by virtue of which sheep exhibits multiple lambing phenomena. This mutation is now being intensively studied and widely used. However, such effect of the FecB mutation has not been applied to goats, and similar types of the FecB gene in goats still need to be studied. Thus, the current study attempted to verify potential mutations in the goat FecB gene as well as investigate their functions related to fecundity. First, FecB expression was investigated in six different goat tissues, and we found that FecB expression was highest in the mammary gland, followed by the ovary. Next, the influence of the FecB gene was analyzed from a new perspective, where five potential copy number variations (CNVs) (CNV1–5) within the FecB gene were identified for the first time, and then their effects on litter size were measured. Our results point out that CNV3 (P = 3.44E-4) and CNV5 (P = 0.034) could significantly influence the litter size of goats. Identically, the combination genotype of CNV3 and CNV5 which consisted of their dominant genotypes was also significantly associated with goat litter size (P = 7.80E-5). Hence, CNV3 and CNV5 could serve as potential DNA molecular markers applied to DNA editing and DNA microarray. Additionally, the abovementioned study has laid a theoretical foundation for the detection of potential fertility-related quantitative trait loci within the goat FecB gene.

A novel m6A-related prognostic signature for predicting the overall survival of hepatocellular carcinoma patients.

Liver hepatocellular carcinoma (LIHC) comprises most cases of liver cancer with a poor prognosis. N 6‐methyladenosine (m6A) plays important biological functions in cancers. Thus, the present research was aimed to determine biomarkers of m6A regulators that could effectively predict the prognosis of LIHC patients. Based on the data collected from the Cancer Genome Atlas (TCGA) database, the correlation between the mRNA expression levels and copy number variation (CNV) patterns were determined. Higher mRNA expression resulted from the increasing number of 9 genes. Using the univariate Cox regression analysis, 11 m6A regulators that had close correlations with the LIHC prognosis were identified. In addition, under the support of the multivariate Cox regression models and the least absolute shrinkage and selection operator, a 4‐gene (YTHDF2, IGF2BP3, KIAA1429, and ALKBH5) signature of m6A regulators was constructed. This signature was expected to present a prognostic value in LIHC (log‐rank test p value < 0.0001). The GSE76427 (n = 94) and ICGC‐LIRI‐JP (n = 212) datasets were used to validate the prognostic signature, suggesting strong power to predict patients' prognosis for LIHC. To sum up, genetic alterations in m6A regulatory genes were identified as reliable and effective biomarkers for predicting the prognosis of LIHC patients.

Emergent correlations in gene expression dynamics as footprints of resource competition.

Genetic circuits need a cellular environment to operate in, which naturally couples the circuit function with the overall functionality of gene regulatory network. To execute their functions, all gene circuits draw resources in the form of RNA polymerases, ribosomes, and tRNAs. Recent experiments pointed out that the role of resource competition on synthetic circuit outputs could be immense. However, the effect of complexity of the circuit architecture on resource sharing dynamics is yet unexplored. In this paper, we employ mathematical modelling and in-silico experiments to identify the sources of resource trade-off and to quantify its impact on the function of a genetic circuit, keeping our focus on regulation of immediate downstream proteins, which are often used as protein read-outs. We show that estimating gene expression dynamics from readings of downstream protein data might be unreliable when the resource is limited and ribosome affinities are asymmetric. We focus on the impact of mRNA copy number and ribosome binding site (RBS) strength on the nonlinear isocline that emerges with two regimes, prominently separated by a tipping point, and study how correlation and competition dominate each other depending on various circuit parameters. Focusing further on genetic toggle circuit, we have identified major effects of resource competition in this model motif and quantified the observations. The observations are testable in wet-lab experiments, as all the parameters chosen are experimentally relevant.

Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5′ leader region, which usually contains the Shine–Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin–antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine–Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine–Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine–Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine–Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine–Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

Exercise-Induced Lactate Release Mediates Mitochondrial Biogenesis in the Hippocampus of Mice via Monocarboxylate Transporters.

Regular exercise training induces mitochondrial biogenesis in the brain via activation of peroxisome proliferator-activated receptor gamma-coactivator 1α (PGC-1α). However, it remains unclear whether a single bout of exercise would increase mitochondrial biogenesis in the brain. Therefore, we first investigated whether mitochondrial biogenesis in the hippocampus is affected by a single bout of exercise in mice. A single bout of high-intensity exercise, but not low- or moderate-intensity, increased hippocampal PGC-1α mRNA and mitochondrial DNA (mtDNA) copy number at 12 and 48h. These results depended on exercise intensity, and blood lactate levels observed immediately after exercise. As lactate induces mitochondrial biogenesis in the brain, we examined the effects of acute lactate administration on blood and hippocampal extracellular lactate concentration by in vivo microdialysis. Intraperitoneal (I.P.) lactate injection increased hippocampal extracellular lactate concentration to the same as blood lactate level, promoting PGC-1α mRNA expression in the hippocampus. However, this was suppressed by administering UK5099, a lactate transporter inhibitor, before lactate injection. I.P. UK5099 administration did not affect running performance and blood lactate concentration immediately after exercise but attenuated exercise-induced hippocampal PGC-1α mRNA and mtDNA copy number. In addition, hippocampal monocarboxylate transporters (MCT)1, MCT2, and brain-derived neurotrophic factor (BDNF) mRNA expression, except MCT4, also increased after high-intensity exercise, which was abolished by UK5099 administration. Further, injection of 1,4-dideoxy-1,4-imino-D-arabinitol (glycogen phosphorylase inhibitor) into the hippocampus before high-intensity exercise suppressed glycogen consumption during exercise, but hippocampal lactate, PGC-1α, MCT1, and MCT2 mRNA concentrations were not altered after exercise. These results indicate that the increased blood lactate released from skeletal muscle may induce hippocampal mitochondrial biogenesis and BDNF expression by inducing MCT expression in mice, especially during short-term high-intensity exercise. Thus, a single bout of exercise above the lactate threshold could provide an effective strategy for increasing mitochondrial biogenesis in the hippocampus.

Urinary cell mRNA profiling of kidney allograft recipients: A systematic investigation of a filtration based protocol for the simplification of urine processing

BackgroundKidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality. MethodsWe isolated RNA from urine collected from kidney allograft recipients using the Cornell centrifugation-based protocol (CCBP) or the Zymo filter-based protocol (ZFBP) and compared RNA purity and yield using a spectrophotometer or a fluorometer and measured absolute copy number of transcripts using customized real-time quantitative PCR assays. We investigated the performance characteristics of RNA isolated using ZFBP and stored either at -80 °C or at ambient temperature for 2 to 4 days and also when shipped to our Gene Expression Monitoring (GEM) Core at ambient temperature. We examined the feasibility of initial processing of urine samples by kidney allograft recipients trained by the GEM Core staff and the diagnostic utility for acute rejection, of urine processed using the ZFBP. ResultsRNA purity (P = 0.0007, Wilcoxon matched paired signed-ranks test) and yield (P < 0.0001) were higher with ZFBP vs. CCBP, and absolute copy number of 18S rRNA was similar (P = 0.79) following normalization of RNA yield by reverse transcribing a constant amount of RNA isolated using either protocol. RNA purity, yield, and absolute copy numbers of 18S rRNA, TGF-β1 mRNA and microRNA-26a were not different (P > 0.05) in the filtrates containing RNA stored either at -80 °C or at ambient temperature for 2 to 4 days or shipped overnight at ambient temperature. RNA purity, yield, and absolute copy numbers of 18S rRNA and TGF-β1 mRNA were also not different (P > 0.05) between home processed and laboratory processed urine filtrates. Urinary cell levels of mRNA for granzyme B (P = 0.01) and perforin (P = 0.0002) in the filtrates were diagnostic of acute rejection in human kidney allografts. ConclusionsUrinary cell mRNA profiling was simplified using the ZFBP without undermining RNA quality or diagnostic utility. Home processing by the kidney allograft recipients, the stability of RNA containing filtrates at ambient temperature, and the elimination of the need for centrifuges and freezers represent some of the advantages of ZFBP over the CCBP for urinary cell mRNA profiling.

Oestrogen-regulated protein SLC39A6: a biomarker of good prognosis in luminal breast cancer

PurposeThe outcome of the luminal oestrogen receptor-positive (ER +) subtype of breast cancer (BC) is highly variable and patient stratification needs to be refined. We assessed the prognostic significance of oestrogen-regulated solute carrier family 39 member 6 (SLC39A6) in BC, with emphasis on ER + tumours.Materials and methodsSLC39A6 mRNA expression and copy number alterations were assessed using the METABRIC cohort (n = 1980). SLC39A6 protein expression was evaluated in a large (n = 670) and annotated series of early-stage (I–III) operable BC using tissue microarrays and immunohistochemistry. The associations between SLC39A6 expression and clinicopathological parameters, patient outcomes and other ER-related markers were evaluated using Chi-square tests and Kaplan–Meier curves.ResultsHigh SLC39A6 mRNA and protein expression was associated with features characteristic of less aggressive tumours in the entire BC cohort and ER + subgroup. SLC39A6 protein expression was detected in the cytoplasm and nuclei of the tumour cells. High SLC39A6 nuclear expression and mRNA levels were positively associated with ER + tumours and expression of ER-related markers, including the progesterone receptor, forkhead box protein A1 and GATA binding protein 3. In the ER + luminal BC, high SLC39A6 expression was independently associated with longer BC-specific survival (BCSS) (P = 0.015, HR 0.678, 95% CI 0.472‒0.972) even in those who did not receive endocrine therapy (P = 0.001, HR 0.701, 95% CI 0.463‒1.062).ConclusionSLC39A6 may be prognostic for a better outcome in ER + luminal BC. Further functional studies to investigate the role of SLC39A6 in ER + luminal BC are warranted.

Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11β-HSD2-Related Hypertension.

ObjectiveApparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH).Aim of the StudyTo detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension.MethodsIn this observational case–control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (β-2 microglobulin) gene was selected as the reference housekeeping gene.ResultsAmong family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118–220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92–124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8–38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5–14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression.ConclusionsHSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Using Glycerol to Produce European Sea Bass Feed With Oleaginous Microbial Biomass: Effects on Growth Performance, Filet Fatty Acid Profile, and FADS2 Gene Expression

Using a circular economy concept, the present study investigated the use of crude glycerol, a primary by-product of biodiesel production, as a low-priced nutrient source for heterotrophic cultivation of the fungus-like protist Schizochytrium limacinum SR21 strain. The whole biomass of this oleaginous microorganism, rich in docosahexaenoic acid (DHA) and high-quality proteins, was then paired with a vegetable oil (VO) source and used to replace fish oil (FO) in European sea bass (Dicentrarchus labrax) feeds. Four nutritionally balanced diets were formulated: diet FO (a FO-based diet), diet VO + 0 (a VO-based diet without S. limacinum), and diets VO + 5 and VO + 10 that were VO-based feeds supplemented with 5 and 10% of S. limacinum, respectively. After a 3-month feeding trial, fish of all dietary groups tripled their initial weight, but growth and feeding efficiencies of D. labrax were not significantly different among treatments. Although the formulated diets were balanced for polyunsaturated fatty acids (PUFAs), fish fed with feeds containing either VO or VO plus 5 and 10% of S. limacinum biomass had significantly higher levels of PUFAs in the flesh than fish fed the FO-based diet. Values of health-related lipid indexes, such as atherogenicity index, thrombogenicity index, and flesh lipid quality as well as n-6/n-3 and PUFAs/SFAs ratios confirmed the high nutritional value of sea bass filet, thus representing a healthy product for human consumption. Although the PUFAs/SFAs ratio showed a significantly higher value in fish fed with VO-based diets supplemented with S. limacinum than in those fed with FO diet, suggesting a better filet quality, the n-6/n-3 ratio clearly indicated that filet quality of dietary group FO was best (value of 0.55) and that of group VO + 10 second best (value of 0.98). We also evaluated the nutritional regulation of Δ6-desaturase (or fads2) gene expression in European sea bass liver. European sea bass fed the VO + 0 diet had the highest number of mRNA copies for Δ6-desaturase (or fads2), fish fed with diet VO + 10 the lowest. Our study adds to the growing body of literature concerning the use of thraustochytrid biomass as a valid alternative to marine-derived raw materials for European sea bass feeds.

The Sex Differences in Uveal Melanoma: Potential Roles of EIF1AX, Immune Response and Redox Regulation.

Background: Uveal melanoma (UVM) is a rare cancer that shows sex difference in incidence and survival, with little previous report for the underlying mechanism. Methods: This study used the SEER data (1974–2016) for an age-dependent analysis on sex difference in UVM, and further used the TCGA-UVM genomics dataset for analyzing the differential gene expression profiles in tumors from men and women. Results: Our results demonstrate a sex difference in older age (≥40 years) but not in younger patients, with men exhibiting a higher incidence rate than women. However, younger women have shown a continuous increasing trend since 1974. Examining the 11 major oncogenes and tumor suppressors in UVM revealed that EIF1AX showed a significant sex difference in mRNA accumulation and copy number variation, with female tumors expressing higher levels of EIF1AX and exhibiting more variations in copy numbers. EIF1AX mRNA levels were significantly inversely correlated with EIF1AX copy numbers in female tumors only, but not in male tumors. Differential gene expression analysis at the whole genomic level identified a set of 92 protein-coding and 16 RNA-coding genes which exhibited differential expression in men and women (fold of change cutoff at 1.7, adjusted p value < 0.05, FDR < 0.05). Network analysis showed significant difference in immune response and in disulfide bond formation, with EGR1/EGR2 and PDIA2 genes as regulators for immune response and disulfide bond formation, respectively. The melanocortin pathway which is linked to both melanin synthesis and obesity seems to be altered with unclear significance, as the sex difference in POMC, DCT/TYRP2, and MRAP2 was observed but with no clear direction. Conclusion: This study reveals possible mechanisms for the sex difference in tumorigenesis of UVM which has potentials for better understanding and prevention of UVM.

Prostaglandin EP4 receptor mRNA expression in canine lymphoma.

Canine lymphoma (LSA) is a diverse, aggressive malignancy initiated by a variety of factors. Understanding those factors could help identify potential treatment options. Chronic inflammation drives lymphoma in human medicine and is suspected to play a role in veterinary medicine. The exact mechanisms, however, have not been elucidated. Upregulation of the cyclooxygenase enzymes, and subsequently prostaglandins, potentially play a stimulatory role. Prostaglandins work through one of four EP receptors (EP1-EP4) and the effects mediated through EP4R specifically are thought to be the primary drivers of cancer development. In human T-cell LSA, overexpression of EP4R has been found and appears to protect LSA cells from apoptosis. The role of EP4R in human B-cell LSA is more nuanced. This study aims to evaluate the mRNA expression of the EP4R gene (ptger4) in canine B-cell and T-cell LSA. Archived canine lymph nodes with histologically confirmed B-cell and T-cell LSA, and reactive lymph nodes, were evaluated for EP4R mRNA expression using a novel RNA in situ hybridization technique (RNAscope). Quantification of RNAscope signals was completed with an advanced digital pathology image analysis system (HALO). Results were reported as copy number, H-score, and percent tumour cell expression of EP4R mRNA. All reactive, B-cell LSA, and T-cell LSA lymph nodes expressed EP4R mRNA. The mRNA copy number, H-score, and percent tumour cell expression of EP4R were higher in B-cell (p < .003) and T-cell (p < .001) LSA samples compared to reactive lymph node samples. There were no differences between B-cell LSA and T-cell LSA.

A Moderate Increase in the WT1 mRNA Copy Number in Peripheral Blood of Patients with Long-Standing Acquired Aplastic Anemia without Apparent Signs of Myelodysplastic Syndrome

Background: The copy number of Wilms tumor 1 mRNA (WT1) correlates well with the acute myeloid leukemia (AML) cell burden and is being used as a reliable marker of residual AML cells. WT1 has also been shown to be useful for distinguishing high-risk myelodysplastic syndrome from low-risk myelodysplastic syndrome (MDS) as well as diagnosing disease progression with time in patients with low-risk MDS. However, few studies have so far focused on the role of measuring WT1 in diagnosing clonal evolution in patients with acquired aplastic anemia (AA). Although normal values of WT1 are defined as <50 copies/µgRNA, the reference values in AA patients without a propensity toward progression to MDS also remain unclear. To address these issues, we retrospectively studied 36 patients with bone marrow failure whose WT1 copy numbers in peripheral blood (PB) were serially measured to determine their correlation with the clinical picture and prognosis of the patients. Patients & Methods: PB samples of 36 patients (male/female, 19/17; a median age, 53.5 [range, 23-85] years) were subjected to WT1 measurement using a quantitative PCR method. The diagnoses of the patients were acquired AA in 29, MDS-multilineage dysplasia (MLD) evolved from AA in 4, and hemolytic paroxysmal nocturnal hemoglobinuria (PNH) in 3. Among 29 AA patients, 2 was untreated, and 22 were in convalescence while 5 were refractory after 0.5-457 months of immunosuppressive therapy (IST) and/or androgen therapy. WT1 was serially measured over time for all the patients except 5 whose initial examination produced negative (<50 copies/µgRNA) results. Myeloid malignancy-related gene abnormalities were screened in 11 patients showing an increase in the WT1 copy number. Results: WT1 at the initial examination was negative in 12 and positive in 17 (59%) patients with AA ranging from 59 to 520 copies. The 17 patients with increased WT1 had a disease duration of 122 months (11-495) but did not shown any signs of progression to MDS. The WT1 levels in patients possessing PNH-type cells (<50-330, median 62) were comparable to those without PNH-type cells (<67-520, median 89. There was a significant difference in the WT1 level between the 10 patients with a disease duration <3 years (<50-190, median <50) and the 19 patients with a disease duration ≥3 years (<50-520, median, 110, P=0.032). All 6 patients with WT1>200 had a disease duration ≥3 years. In contrast, the WT1 levels in the 4 patients who relapsed with cytopenia due to progression to MDS were as high as 690-5,700 (median, 2,400). Deep sequencing revealed somatic mutations of myeloid malignancy-related genes in 4 of 11 patients studied, which included EZH2 in an MDS patient (WT1 2,000), BCOR and DNMT3A in another MDS patient (WT1 2,800), and ASXL1 in a PNH patients (WT1 96). Conclusions: The WT1 copy numbers in PB of AA patients were higher than in AML patients with minimal residual diseases, and the cut-off value that helps diagnose MDS evolving from AA was considered to be around 600 copies. The high WT1 levels in long-standing AA patients without any signs of progression to MDS may represent an increased proliferative potential of hematopoietic stem/progenitor cells that support sustained hematopoiesis with their limited numbers. An increase in the WT1 copy number was associated with somatic mutations in some MDS patients but not in AA patients. Although AA patients with increased WT1 copy numbers need to be carefully followed, a moderate increase in the WT1 copy number does not portend myeloid malignancies unless the number steadily increases. DisclosuresIshiyama:Alexion Pharma GK: Research Funding.

Identification of genetic variations associated with drug resistance in non-small cell lung cancer patients undergoing systemic treatment

Non-small cell lung cancer (NSCLC) is characterized by relatively rapid response to systemic treatments yet inevitable resistance and predisposed to distant metastasis. We thus aimed at performing sequencing analysis to determine genomic events and underlying mechanisms concerning drug resistance in NSCLC. We performed targeted sequencing of 40 medication-relevant genes on plasma samples from 98 NSCLC patients and analyzed impact of genetic alterations on clinical presentation as well as response to systemic treatments. Profiling of multi-omics data from 1024 NSCLC tissues in public datasets was carried out for comparison and validation of identified molecular events implicated in resistance. A genetic association of CYP2D6 deletion with drug resistance was identified through circulating tumor DNA (ctDNA) profiling and response assessment. FCGR3A amplification was potentially involved in resistance to EGFR inhibitors. We further verified our findings in tissue samples and focused on potential resistance mechanisms, which uncovered that depleted CYP2D6 affected a set of genes involved in EMT, oncogenic signaling as well as inflammatory pathways. Tumor microenvironment analysis revealed that NSCLC with CYP2D6 loss manifested increased levels of immunomodulatory gene expressions, PD-L1 expression, relatively high mutational burden and lymphocyte infiltration. DNA methylation alterations were also found to be correlated with mRNA expressions and copy numbers of CYP2D6. Finally, MEK inhibitors were identified by CMap as the prospective therapeutic drugs for CYP2D6 deletion. These analyses identified novel resistance mechanisms to systemic NSCLC treatments and had significant implications for the development of new treatment strategies.

Frequency Domain Analysis of Fluctuations of mRNA and Protein Copy Numbers within a Cell Lineage: Theory and Experimental Validation

The stochasticity of gene expression is manifested in the fluctuations of messenger ribonucleic acid and protein copy numbers within a cell lineage over time. While data of this type can be obtained for many generations, most mathematical models are unsuitable to interpret such data since they assume nongrowing cells. Here we develop a theoretical approach that quantitatively links the frequency content of lineage data to subcellular dynamics. We elucidate how the position, height, and width of the peaks in the power spectrum provide a distinctive fingerprint that encodes a wealth of information about mechanisms controlling transcription, translation, replication, degradation, bursting, promoter switching, cell cycle duration, cell division, gene dosage compensation, and cell-size homeostasis. Predictions are confirmed by analysis of single-cell Escherichia coli data obtained using fluorescence microscopy. Furthermore, by matching the experimental and theoretical power spectra, we infer the temperature-dependent gene expression parameters, without the need of measurements relating fluorescence intensities to molecule numbers.Received 13 October 2020Revised 8 February 2021Accepted 8 March 2021DOI:https://doi.org/10.1103/PhysRevX.11.021032Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.Published by the American Physical SocietyPhysics Subject Headings (PhySH)Research AreasFluctuationsGene expressionNonequilibrium fluctuationsTechniquesMaster equationInterdisciplinary PhysicsStatistical PhysicsBiological Physics

Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells.

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasma pneumoniae, Helicobacter pylori, Fusobacterium nucleatum, Chlamydia thrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.

The association between the expression of PD-L1 and CMTM6 in undifferentiated pleomorphic sarcoma.

Undifferentiated pleomorphic sarcoma (UPS) is a sarcoma with a poor prognosis. A clinical trial, SARC028, revealed that treatment with anti-PD-1 drugs was effective against UPS. Studies have reported that UPS expresses PD-L1, sometimes strongly (≥ 50%). However, the mechanism of PD-L1 expression in UPS has remained unclear. CKLF-like MARVEL transmembrane domain containing 6 (CMTM6) was identified as a novel regulator of PD-L1 expression. The positive relationship between PD-L1 and CMTM6 has been reported in several studies. The aim of this study was thus to examine CMTM6 expression in UPS and evaluate the relationship between PD-L1 and CMTM6 in this disease. Fifty-one primary UPS samples were subjected to CMTM6 and PD-L1 immunostaining. CMTM6 expression was assessed using proportion and intensity scores. CMTM6 gene copy number was also evaluated using a real-time PCR-based copy number assay. We also analyzed the mRNA expression and copy number variation of PD-L1 and CMTM6 in The Cancer Genome Atlas (TCGA) data. TCGA data indicated that the mRNAs encoded by genes located around 3p22 were coexpressed with CMTM6 mRNA in UPS. Both proportion and intensity scores of CMTM6 positively correlated with strong PD-L1 expression (≥ 50%) (both p = 0.023). CMTM6 copy number gain increased CMTM6 expression. Patients with UPS with a high CMTM6 intensity score had a worse prognosis for overall survival. UPS showed variation in CMTM6 copy number and CMTM6 expression. CMTM6 expression was significantly correlated with PD-L1 expression, especially with strong PD-L1 expression.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) is an arterivirus newly discovered in Chinese softshell turtles. Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles. In this study, a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system, and its polyclonal antibody was generated. The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay (dot-ELISA). The distribution of TSHSV in different organs of T. sinensis was examined by immunohistochemistry (IHC) and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed, and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles. The IHC assay indicated that the virus was highly localized in various cells, including intestinal lymphocytes, enterocytes, kidney epithelial cells, spleen cells, lung macrophages, and cardiomyocytes. The qRT-PCR analysis revealed that TSHSV was detected in all organs tested, including the lungs, liver, kidneys, spleen, and heart. The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group. The interferon-stimulated genes (ISGs), myxovirus resistance protein 2 (MX2) and radical S-adenosyl methionine domain containing 2 (RSAD2) were highly upregulated in all groups of infected turtles. Antibody-dependent enhancement (ADE) seemed to occur after stimulation by the polyclonal antibody, because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group. Overall, these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression.

The HLA gene complex is the most important single genetic factor in susceptibility to most diseases with autoimmune or autoinflammatory origin and in transplantation matching. Most studies have focused on the vast allelic variation in these genes; only a few studies have explored differences in the expression levels of HLA alleles. In this study, we quantified mRNA expression levels of HLA class I and II genes from peripheral blood samples of 50 healthy individuals. The gene- and allele-specific mRNA expression was assessed using unique molecular identifiers, which enabled PCR bias removal and calculation of the number of original mRNA transcripts. We identified differences in mRNA expression between different HLA genes and alleles. Our results suggest that HLA alleles are differentially expressed and these differences in expression levels are quantifiable using RNA sequencing technology. Our method provides novel insights into HLA research, and it can be applied to quantify expression differences of HLA alleles in various tissues and to evaluate the role of this type of variation in transplantation matching and susceptibility to autoimmune diseases.

Impact of between-tissue differences on pan-cancer predictions of drug sensitivity.

Increased availability of drug response and genomics data for many tumor cell lines has accelerated the development of pan-cancer prediction models of drug response. However, it is unclear how much between-tissue differences in drug response and molecular characteristics may contribute to pan-cancer predictions. Also unknown is whether the performance of pan-cancer models could vary by cancer type. Here, we built a series of pan-cancer models using two datasets containing 346 and 504 cell lines, each with MEK inhibitor (MEKi) response and mRNA expression, point mutation, and copy number variation data, and found that, while the tissue-level drug responses are accurately predicted (between-tissue ρ = 0.88-0.98), only 5 of 10 cancer types showed successful within-tissue prediction performance (within-tissue ρ = 0.11-0.64). Between-tissue differences make substantial contributions to the performance of pan-cancer MEKi response predictions, as exclusion of between-tissue signals leads to a decrease in Spearman's ρ from a range of 0.43-0.62 to 0.30-0.51. In practice, joint analysis of multiple cancer types usually has a larger sample size, hence greater power, than for one cancer type; and we observe that higher accuracy of pan-cancer prediction of MEKi response is almost entirely due to the sample size advantage. Success of pan-cancer prediction reveals how drug response in different cancers may invoke shared regulatory mechanisms despite tissue-specific routes of oncogenesis, yet predictions in different cancer types require flexible incorporation of between-cancer and within-cancer signals. As most datasets in genome sciences contain multiple levels of heterogeneity, careful parsing of group characteristics and within-group, individual variation is essential when making robust inference.