Gene Expression Profile Of Markers Research Articles

Unfolding the ventral nerve center of chaetognaths

BackgroundChaetognaths are a clade of marine worm-like invertebrates with a heavily debated phylogenetic position. Their nervous system superficially resembles the protostome type, however, knowledge regarding the molecular processes involved in neurogenesis is lacking. To better understand these processes, we examined the expression profiles of marker genes involved in bilaterian neurogenesis during post-embryonic stages of Spadella cephaloptera. We also investigated whether the transcription factor encoding genes involved in neural patterning are regionally expressed in a staggered fashion along the mediolateral axis of the nerve cord as it has been previously demonstrated in selected vertebrate, insect, and annelid models.MethodsThe expression patterns of genes involved in neural differentiation (elav), neural patterning (foxA, nkx2.2, pax6, pax3/7, and msx), and neuronal function (ChAT and VAChT) were examined in S. cephaloptera hatchlings and early juveniles using whole-mount fluorescent in situ hybridization and confocal microscopy.ResultsThe Sce-elav+ profile of S. cephaloptera hatchlings reveals that, within 24 h of post-embryonic development, the developing neural territories are not limited to the regions previously ascribed to the cerebral ganglion, the ventral nerve center (VNC), and the sensory organs, but also extend to previously unreported CNS domains that likely contribute to the ventral cephalic ganglia. In general, the neural patterning genes are expressed in distinct neural subpopulations of the cerebral ganglion and the VNC in hatchlings, eventually becoming broadly expressed with reduced intensity throughout the CNS in early juveniles. Neural patterning gene expression domains are also present outside the CNS, including the digestive tract and sensory organs. ChAT and VAChT domains within the CNS are predominantly observed in specific subpopulations of the VNC territory adjacent to the ventral longitudinal muscles in hatchlings.ConclusionsThe observed spatial expression domains of bilaterian neural marker gene homologs in S. cephaloptera suggest evolutionarily conserved roles in neurogenesis for these genes among bilaterians. Patterning genes expressed in distinct regions of the VNC do not show a staggered medial-to-lateral expression profile directly superimposable to other bilaterian models. Only when the VNC is conceptually laterally unfolded from the longitudinal muscle into a flat structure, an expression pattern bearing resemblance to the proposed conserved bilaterian mediolateral regionalization becomes noticeable. This finding supports the idea of an ancestral mediolateral patterning of the trunk nervous system in bilaterians.

Atp6i deficient mouse model uncovers transforming growth factor-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i−/−) arrested tooth root formation, indicated by truncated Hertwig’s epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i−/− mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i−/− mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-β (TGF-β) signaling in Atp6i−/− odontoblasts. Through treating pulp precursor cells with Atp6i−/− or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-β1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i−/− OC conditioned medium. Importantly, ectopic TGF-β1 partially rescued root development and root dentin deposition of Atp6i−/− mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-β1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-β1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.

The expression profiles of signature genes from CD103+LAG3+ tumour-infiltrating lymphocyte subsets predict breast cancer survival.

Tumour-infiltrating lymphocytes (TILs), including T and B cells, have been demonstrated to be associated with tumour progression. However, the different subpopulations of TILs and their roles in breast cancer remain poorly understood. Large-scale analysis using multiomics data could uncover potential mechanisms and provide promising biomarkers for predicting immunotherapy response. Single-cell transcriptome data for breast cancer samples were analysed to identify unique TIL subsets. Based on the expression profiles of marker genes in these subsets, a TIL-related prognostic model was developed by univariate and multivariate Cox analyses and LASSO regression for the TCGA training cohort containing 1089 breast cancer patients. Multiplex immunohistochemistry was used to confirm the presence of TIL subsets in breast cancer samples. The model was validated with a large-scale transcriptomic dataset for 3619 breast cancer patients, including the METABRIC cohort, six chemotherapy transcriptomic cohorts, and two immunotherapy transcriptomic cohorts. We identified two TIL subsets with high expression of CD103 and LAG3 (CD103+LAG3+), including a CD8+ T-cell subset and a B-cell subset. Based on the expression profiles of marker genes in these two subpopulations, we further developed a CD103+LAG3+ TIL-related prognostic model (CLTRP) based on CXCL13 and BIRC3 genes for predicting the prognosis of breast cancer patients. CLTRP-low patients had a better prognosis than CLTRP-high patients. The comprehensive results showed that a low CLTRP score was associated with a high TP53 mutation rate, high infiltration of CD8 T cells, helper T cells, and CD4 T cells, high sensitivity to chemotherapeutic drugs, and a good response to immunotherapy. In contrast, a high CLTRP score was correlated with a low TP53 mutation rate, high infiltration of M0 and M2 macrophages, low sensitivity to chemotherapeutic drugs, and a poor response to immunotherapy. Our present study showed that the CLTRP score is a promising biomarker for distinguishing prognosis, drug sensitivity, molecular and immune characteristics, and immunotherapy outcomes in breast cancer patients. The CLTRP could serve as a valuable tool for clinical decision making regarding immunotherapy.

C-type natriuretic peptide (CNP) inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor growth by modulating inflammation in Swiss albino mice.

C-type natriuretic peptide (CNP) exhibits anti-inflammatory activity besides its natriuretic and diuretic functions. The present study aimed to determine the anticancer and synergistic therapeutic activity of CNP against a 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor mouse model. CNP (2.5 µg/kg body weight) was injected either alone and/or in combination with Cisplatin (CDDP) (2 mg/kg body weight) for 4 weeks. The dorsal skin tumor incidences/growth and mortality rate were recorded during the experimental period of 16 weeks. The serum C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels, infiltrating mast cells, and AgNORs proliferating cells count were analyzed in control and experimental mice. Further, the expression profile of marker genes of proliferation, inflammation, and progression molecules were analyzed using Reverse transcriptase-polymerase chain reaction(RT-PCR)/quantitative PCR(qPCR), western blot, and immunohistochemistry. The DMBA/Croton oil-induced mice exhibited 100% tumor incidence. Whereas, CNP alone, CDDP alone, and CNP+CDDP combination-treated mice exhibited 58%, 46%, and 24% tumor incidence, respectively. Also, a marked reduction in the levels of serum CRP and LDH, the number of infiltrating mast cells count and AgNORs proliferating cells count were noticed in the mice skin sections. Further, a significant reduction in both mRNA and protein expression levels of proliferation, inflammation, and progression markers were noticed in CNP (p < 0.01), CDDP (p < 0.01), and CNP+CDDP combination (p < 0.001) treated mice, respectively. The results of the present study suggest that CNP has anticancer activity. Further, the CNP+CDDP treatment has more promising anticancer activity as compared with CNP or CDDP alone treatment, probably due to the synergistic antiproliferative and anti-inflammatory activities of CNP and CDDP.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, Tcell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific Tcells was inhibited by Sephin1. A special TCR+ macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

3D Disease Modelling of Hard and Soft Cancer Using PHA-Based Scaffolds.

Simple SummaryTumour progression in vivo was able to be well mimicked in 3D culture by utilizing biodegradable 10 mm × 10 mm × 8 mm P(3HO-co-3HD) and P(3HB)-based 3D scaffolds with a pore size of 30 to 300 µm. Both hard (MCF7 and MDA-MB-231) and soft (HCT116) tumour-related cells were successfully grown on the scaffolds, and their growth patterns were studied for 5 days. MDA-MB-231 tend to grow in clusters, and MCF7 cells form an evenly dispersed layer, which covered most of the 3D PHA scaffolds, while HCT116 formed large colonies within the pockets of the 3D PHA scaffold. Epithelial mesenchymal transition (EMT) marker genes, including Wnt-11, E-cadherin, Vim and Snail expression profiles, were like those seen in real tumour samples, which confirmed that the cancer models were exhibiting real tumour-like characteristics with high fidelity. These models are important in mimicking hypoxic tumours and in studying gene expression, cellular signalling, angiogenesis and drug response for translational research.Tumour cells are shown to change shape and lose polarity when they are cultured in 3D, a feature typically associated with tumour progression in vivo, thus making it significant to study cancer cells in an environment that mimics the in vivo milieu. In this study we established hard (MCF7 and MDA-MB-231, breast cancer) and soft (HCT116, colon cancer) 3D cancer tumour models utilizing a blend of P(3HO-co-3HD) and P(3HB). P(3HO-co-3HD) and P(3HB) belong to a group of natural biodegradable polyesters, PHAs, that are synthesised by microorganisms. The 3D PHA scaffolds produced, with a pore size of 30 to 300 µm, allow for nutrients to diffuse within the scaffold and provide the cells with the flexibility to distribute evenly within the scaffold and grow within the pores. Interestingly, by Day 5, MDA-MB-231 showed dispersed growth in clusters, and MCF7 cells formed an evenly dispersed dense layer, while HCT116 formed large colonies within the pockets of the 3D PHA scaffolds. Our results show Epithelial Mesenchymal Transition (EMT) marker gene expression profiles in the hard tumour cancer models. In the 3D-based PHA scaffolds, MDA-MB-231 cells expressed higher levels of Wnt-11 and mesenchymal markers, such as Snail and its downstream gene Vim mRNAs, while MCF7 cells exhibited no change in their expression. On the other hand, MCF7 cells exhibited a significantly increased E-Cadherin expression as compared to MDA-MB-231 cells. The expression levels of EMT markers were comparative to their expression reported in the tumour samples, making them good representative of cancer models. In future these models will be helpful in mimicking hypoxic tumours, in studying gene expression, cellular signalling, angiogenesis and drug response more accurately than 2D and perhaps other 3D models.

Gene Expression Analysis of Potato (Solanum tuberosum L.) Lipoxygenase Cascade and Oxylipin Signature under Abiotic Stress.

The metabolism of polyunsaturated fatty acids through the lipoxygenase-catalyzed step and subsequent reactions is referred to as the lipoxygenase (LOX) pathway. The components of this system, such as jasmonates, are involved in growth, development and defense reactions of plants. In this report, we focus on dynamics of expression of different LOX pathway genes and activities of target enzymes with three abiotic stress factors: darkness, salinity and herbicide toxicity. To obtain a more complete picture, the expression profiles of marker genes for salicylic acid, abscisic acid, ethylene, auxin and gibberellin-dependent signaling systems under the same stresses were also analyzed. The gene expression in Solanum tuberosum plants was analyzed using qRT-PCR, and we found that the LOX-cascade-related genes responded to darkness, salinity and herbicide toxicity in different ways. We detected activation of a number of 9-LOX pathway genes; however, in contrast to studies associated with biotic stress (infection), the 9-divinyl ether synthase branch of the LOX cascade was inhibited under all three stresses. GC-MS analysis of the oxylipin profiles also showed the main activity of the 9-LOX-cascade-related enzymes after treatment with herbicide and darkness.

Tumor Heterogeneity and Molecular Characteristics of Glioblastoma Revealed by Single-Cell RNA-Seq Data Analysis.

Glioblastoma multiforme (GBM) is the most common infiltrating lethal tumor of the brain. Tumor heterogeneity and the precise characterization of GBM remain challenging, and the disease-specific and effective biomarkers are not available at present. To understand GBM heterogeneity and the disease prognosis mechanism, we carried out a single-cell transcriptome data analysis of 3389 cells from four primary IDH-WT (isocitrate dehydrogenase wild type) glioblastoma patients and compared the characteristic features of the tumor and periphery cells. We observed that the marker gene expression profiles of different cell types and the copy number variations (CNVs) are heterogeneous in the GBM samples. Further, we have identified 94 differentially expressed genes (DEGs) between tumor and periphery cells. We constructed a tissue-specific co-expression network and protein–protein interaction network for the DEGs and identified several hub genes, including CX3CR1, GAPDH, FN1, PDGFRA, HTRA1, ANXA2 THBS1, GFAP, PTN, TNC, and VIM. The DEGs were significantly enriched with proliferation and migration pathways related to glioblastoma. Additionally, we were able to identify the differentiation state of microglia and changes in the transcriptome in the presence of glioblastoma that might support tumor growth. This study provides insights into GBM heterogeneity and suggests novel potential disease-specific biomarkers which could help to identify the therapeutic targets in GBM.

261 Distinct Melanoma Cancer Stem Cell Profile Defines Response to Immune Checkpoint Inhibitors in Melanomas

This study examined the relationship between cancer stem cells (CSCs) and the susceptibility of melanomas to immunotherapy. Immunotherapy has emerged as a pillar of cancer therapy, demonstrating marked survival benefits in patients with melanoma. However, there remains a significant number of patients who fail to respond to treatment. To improve patient outcomes and optimise appropriate drug selection, it is a priority to unravel the mechanisms underlying the emergence of drug resistance. One of the most important key players to have emerged in the development of drug resistance are the CSCs. Transcriptomic profiles of 158 melanoma biopsies from patients treated with anti-programmed cell death 1 ligand-1 (PD-1) monotherapy (nivolumab or pembrolizumab; n = 63) or combined anti-PD-1 and anti-cytotoxic T lymphocyte antigen 4 (CTLA-4; ipilimumab; n = 57) were obtained. Patients were categorised as responders or non-responders using the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, where responders had a complete response (CR), partial response (PR), or stable disease (SD) of greater than 6 months with no disease progression. Samples were analysed for differential gene expression after normalisation using DESeq2 and HTSeq. Gene levels of well-defined CSCs were compared between responders and non-responders. Changes to gene expression levels were also compared pre and post immunotherapy treatment. This analysis identified a unique melanoma CSC marker gene expression profile “signature” that corresponds to a positive response to immune checkpoint inhibitors. This gene expression profile was associated with longer progression-free survival in patients treated with immunotherapy agents. Therefore, we have identified a CSC gene expression profile that can be used to predict patient response to the current available anti-PD-1 or anti-CTLA4 based therapies. Furthermore, this helps with identifying patients in need of other therapeutic strategies.

Distinguishing mode of action of compounds inducing craniofacial malformations in zebrafish embryos to support dose-response modeling in combined exposures

Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network. A selection of gene expression markers was based on these MOAs. Next, these markers were verified by qPCR in zebrafish embryos, after exposure to reference compounds. These were: triazoles for inhibition of retinoic acid (RA) metabolism, AM580 and CD3254 for selective activation of respectively RA-receptor (RAR) and retinoid-X-receptor (RXR), dithiocarbamates for inhibition of lysyl oxidase, TCDD for activation of the aryl-hydrocarbon-receptor (AhR), VPA for inhibition of histone deacetylase (HDAC), and PFOS for activation of peroxisome proliferator-activated receptor-alpha (PPARα). Next, marker gene profiles for these reference compounds were used to map the profiles of test compounds to known MOAs. In this way, 2,4-dinitrophenol matched with the TCDD and RAR profiles, boric acid with RAR, endosulfan with PFOS, fenpropimorph with dithiocarbamates, PCB126 with AhR, and RA with triazoles and RAR profiles. Prochloraz showed no match. Activities of these compounds in ToxCast assays, and in silico analysis of binding affinity to the respective targets showed limited concordance with the marker gene expression profiles, but still confirmed the complex MOA profiles of reference and test compounds. Ultimately, this approach could be used to support modeling of mixture effects based on upfront knowledge of (dis)similarity of MOAs.

Suppression of Arabidopsis Mediator Subunit-Encoding MED18 Confers Broad Resistance Against DNA and RNA Viruses While MED25 Is Required for Virus Defense.

Mediator subunits play key roles in numerous physiological pathways and developmental processes in plants. Arabidopsis Mediator subunits, MED18 and MED25, have previously been shown to modulate disease resistance against fungal and bacterial pathogens through their role in jasmonic acid (JA) signaling. In this study, Arabidopsis mutant plants of the two Mediator subunits, med18 and med25, were tested against three ssRNA viruses and one dsDNA virus belonging to four different families: Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV), Alternanthera mosaic virus (AltMV), and Cucumber mosaic virus (CMV). Although both subunits are utilized in JA signaling, they occupy different positions (Head and Tail domain, respectively) in the Mediator complex and their absence affected virus infection differently. Arabidopsis med18 plants displayed increased resistance to RNA viral infection and a trend against the DNA virus, while med25 mutants displayed increased susceptibility to all viruses tested at 2 and 14 days post inoculations. Defense marker gene expression profiling of mock- and virus-inoculated plants showed that med18 and med25 mutants exhibited an upregulated SA pathway upon virus infection at 2 dpi for all viruses tested. JA signaling was also suppressed in med18 plants after virus infection, independent of which virus infected the plants. The upregulation of SA signaling and suppression of JA signaling in med18 may have led to more targeted oxidative burst and programmed cell death to control viruses. However, the susceptibility exhibited by med25 mutants suggests that other factors, such as a weakened RNAi pathway, might play a role in the observed susceptibility. We conclude that MED18 and MED25 have clear and opposite effects on accumulation of plant viruses. MED18 is required for normal virus infection, while MED25 is important for defense against virus infection. Results from this study provide a better understanding of the role of Mediator subunits during plant-virus interactions, viral disease progression and strategies to develop virus resistant plants.

Guiding neural extensions of PC12 cells on carbon nanotube tracks dielectrophoretically formed in poly(ethylene glycol) dimethacrylate.

The PC12 cell line has been widely used as an in vitro model for studying neuronal differentiation and identifying the factors affecting the process. It has the ability to differentiate in the presence of nerve growth factor (NGF), resulting in neural extensions called dendrites and axons. In this study, first the impact of randomly distributed multi-walled carbon nanotubes (MWCNTs) in poly(ethylene glycol) dimethacrylate (PEGDMA) on PC12 cell differentiation was investigated in terms of neurite length, number of neurite per cell and differentiation marker gene expression profile. Then, dielectrophoretically aligned MWCNTs in PEGDMA was used to guide and support the neuronal differentiation of PC12 cells in the presence of NGF. The method is expected to be useful in revealing the nanotopographical role in fundamental studies and understanding of nanotopographical effects for biomedical applications on nerve regeneration.

An improved advanced fragment analysis-based classification and risk stratification of pediatric acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) contains cytogenetically distinct subtypes that respond differently to cytotoxic drugs. Therefore, subtype classification is important and indispensable in ALL diagnosis. In our previous study, we identified some marker genes in childhood ALL by means of microarray technology and, furthermore, detected the relative expression levels of 57 marker genes and built a comparatively convenient and cost-effective classifier with a prediction accuracy as high as 94% based on the advanced fragment analysis (AFA) technique.MethodsA more convenient improved AFA (iAFA) technique with one-step multiplex RT-PCR and an anti-contamination system was developed to detect 57 marker genes for ALL.ResultsThe iAFA assay is much easier and more convenient to perform than the previous AFA assay and has a prediction accuracy of 95.29% in ALL subtypes. The anti-contamination system could effectively prevent the occurrence of lab DNA contamination. We also showed that marker gene expression profiles in pediatric ALL revealed 2 subgroups with different outcomes. Most ALL patients (95.8%) had a good-risk genetic profile, and only 4.2% of ALL patients had a poor-risk genetic profile, which predicted an event-free survival (EFS) of 93.6 ± 1.3% vs 18.8 ± 9.8% at 5 years, respectively (P < 0.001).ConclusionsCompared to the previous AFA assay, the iAFA technique is more functional, time-saving and labor-saving. It could be a valuable clinical tool for the classification and risk stratification of pediatric ALL patients.

Ectopic expression of GmZAT4, a putative C2H2-type zinc finger protein, enhances PEG and NaCl stress tolerances in Arabidopsis thaliana.

The zinc finger protein (ZFP) transcription factor family plays an important role in regulating plant growth, development, and response to abiotic stress. In this study, we aimed to determine the role of GmZAT4, a C2H2-type transcription factor, in abiotic stress tolerance. The complete coding sequence of the GmZAT4 gene was isolated from soybean root RNA, which shows highest expression level compared with leaf, flower and other tissues. Using multiple sequence alignment and conserved domain analysis, we showed that GmZAT4 is a typical C2H2-type transcription factor which is comprised of two C2H2 domains, including a highly conserved QALGGH motif, and implied the regulation of abiotic stress tolerance in plant. A phylogenetic tree revealed that the soybean GmZAT4 gene clustered with ZAT4 from Glycine soja and AZF1, AZF2, and AZF3 from Arabidopsis thaliana. The mRNA expression levels of GmZAT4 were determined in two soybean cultivars by quantitative reverse transcription (qRT)-PCR and compared. The results showed higher expression (up to 60, 25 and 4 times, respectively) in the drought-tolerant type (Jinda 74) compared to the drought-sensitive soybean cultivar (Jinda 53) following treatment with 18% PEG, 150mM NaCl, or 100µM abscisic acid (ABA). GmZAT4 was ectopically over-expressed in A. thaliana to determine its role in abiotic stress tolerance. GmZAT4 overexpression enhanced the tolerance of A. thaliana to treatment with 20% PEG and 150mM NaCl, and improved the germination rate following treatment with 1µM or 2µM ABA. The expression profiles of marker genes in the ABA signaling pathway, such as RD29A, RD29B, ABI, and RAD, indicated that GmZAT4 enhanced the abiotic stress tolerance of Arabidopsis. These results suggest that the C2H2-type ZFP encoded by GmZAT4 plays an important role in PEG and NaCl stress tolerance and ABA responses in soybean and A. thaliana.

Gene expression profiling toward the next generation safety control of influenza vaccines and adjuvants in Japan

Influenza becomes epidemic worldwide every year, and many individuals receive vaccination annually. Quality control relating to safety and potency of influenza vaccines is important to maintain public confidence. The safety of influenza vaccines has been assessed by clinical trials, and animal safety tests are performed to monitor the consistent quality between vaccines used for clinical trials and marketing; the biological responses in vaccinated animals are evaluated, including changes in body weight and white blood cell count. Animal safety tests have been contributing to the quality relating to the safety of influenza vaccines for decades, but improvements are needed. Although precise mechanisms involving biological changes in animal safety tests have not been fully elucidated, the application of cDNA microarray technology make it possible to reliably identify genes related to biological responses in vaccinated animals. From analysis of the expression profile of >10,000 genes of lung in animals treated with an inactivated whole virion influenza vaccine, we identified 17 marker genes whose expression patterns correlated well to changes in body weight and leukocyte count in vaccinated animals. In influenza HA vaccine-treated animals exhibiting subtle changes in biological responses, a robust expression pattern of marker genes was found. Furthermore, these marker genes could also be used in the evaluation of adjuvanted influenza vaccines. The expression profile of marker genes is expected to be an alternative indicator for safety control of various influenza vaccines conferring high sensitivity and short turnaround time. Thus, gene expression profiling may be a powerful tool for safety control of vaccines in the future.

The anti-myeloma activity of bone morphogenetic protein 2 predominantly relies on the induction of growth arrest and is apoptosis-independent

Multiple myeloma (MM), a malignancy of the bone marrow, is characterized by a pathological increase in antibody-producing plasma cells and an increase in immunoglobulins (plasmacytosis). In recent years, bone morphogenetic proteins (BMPs) have been reported to be activators of apoptotic cell death in neoplastic B cells in MM. Here, we use bone morphogenetic protein 2 (BMP2) to show that the "apoptotic" effect of BMPs on human neoplastic B cells is dominated by anti-proliferative activities and cell cycle arrest and is apoptosis-independent. The anti-proliferative effect of BMP2 was analysed in the human cell lines KMS12-BM and L363 using WST-1 and a Coulter counter and was confirmed using CytoTox assays with established inhibitors of programmed cell death (zVAD-fmk and necrostatin-1). Furthermore, apoptotic activity was compared in both cell lines employing western blot analysis for caspase 3 and 8 in cells treated with BMP2 and FasL. Additionally, expression profiles of marker genes of different cell death pathways were analysed in both cell lines after stimulation with BMP2 for 48h using an RT-PCR-based array. In our experiments we observed that there was rather no reduction in absolute cell number, but cells stopped proliferating following treatment with BMP2 instead. The time frame (48–72 h) after BMP2 treatment at which a reduction in cell number is detectable is too long to indicate a directly BMP2-triggered apoptosis. Moreover, in comparison to robust apoptosis induced by the approved apoptotic factor FasL, BMP2 only marginally induced cell death. Consistently, neither the known inhibitor of apoptotic cell death zVAD-fmk nor the necroptosis inhibitor necrostatin-1 was able to rescue myeloma cell growth in the presence of BMP2.

Endovascular Biopsy: In Vivo Cerebral Aneurysm Endothelial Cell Sampling and Gene Expression Analysis.

There is limited data describing endothelial cell (EC) gene expression between aneurysms and arteries partly because of risks associated with surgical tissue collection. Endovascular biopsy (EB) is a lower risk alternative to conventional surgical methods, though no such efforts have been attempted for aneurysms. We sought (1) to establish the feasibility of EB to isolate viable ECs by fluorescence-activated cell sorting (FACS), (2) to characterize the differences in gene expression by anatomic location and rupture status using single-cell qPCR, and (3) to demonstrate the utility of unsupervised clustering algorithms to identify cell subpopulations. EB was performed in 10 patients (5 ruptured, 5 non-ruptured). FACS was used to isolate the ECs and single-cell qPCR was used to quantify the expression of 48 genes. Linear mixed models and exploratory multilevel component analysis (MCA) and self-organizing maps (SOMs) were performed to identify possible subpopulations of cells. ECs were collected from all aneurysms and there were no adverse events. A total of 437 ECs was collected, 94 (22%) of which were aneurysmal cells and 319 (73%) demonstrated EC-specific gene expression. Ruptured aneurysm cells, relative controls, yielded a median p value of 0.40 with five genes (10%) with p values <0.05. The five genes (TIE1, ENG, VEGFA, MMP2, and VWF) demonstrated uniformly reduced expression relative the remaining ECs. MCA and SOM analyses identified a population of outlying cells characterized by cell marker gene expression profiles different from endothelial cells. After removal of these cells, no cell clustering based on genetic co-expressivity was found to differentiate aneurysm cells from control cells. Endovascular sampling is a reliable method for cell collection for brain aneurysm gene analysis and may serve as a technique to further vascular molecular research. There is utility in combining mixed and clustering methods, despite no specific subpopulation identified in this trial.

P048 Gene expression profiling of transplant formalin-fixed paraffin-embedded biopsies: comparison of a custom TaqMan® low density array and quantitative nuclease-protection assay

Aim We developed a custom TLDA to monitor changes of gene expression in FFPE renal and small bowel allografts. Here we carried out a parallel comparison of expression profiles of marker genes obtained with TaqMan® technology and with the automated qNPA system. Methods Kidney, small bowel and heart transplant FFPE biopsies with different levels of rejection were selected. 5–6 curls of 10 μm were cut from each biopsy for analysis in TLDA (47 markers including immune, inflammatory and apoptosis genes), and in HTG Edge System using the Immuno-Oncology Expression assay (HTGIOE, 26 markers), or HTG EdgeSeq Oncology Biomarker assay (HTGESOB, 2558 genes, format for NGS). Data was presented as differential expression. Results There are 10 overlapping markers between our TLDA and the HTGIOE assay. Analysis of 30 kidney biopsies with both technologies gave similar results for all 10 markers. Several markers were not detected in some samples analyzed with the HTG Edge System, mostly in normal donor and non-rejection samples. 11 unique markers of HTGIOE assay showed potential value for monitoring gene expression in kidney. 55 samples of heart biopsies were analyzed in TLDA and HTGIOE. Similar results were obtained with both technologies for all overlapping markers. 15 useful markers for monitoring gene expression in heart were found in HTGIOE assay. HTGIOE markers often did not express in non-rejection samples. We also analyzed 17 small bowel biopsies in HTGESOB, which contains 39 of the 47 markers in our TLDA (8 for SB, 17 for both kidney and small bowel, and 14 for kidney). 17.3% of the 2558 genes in the panel showed 2-fold increase expression relative to non-rejection. 15 markers in our TLDA (6 small bowel specific, 7 for both kidney and small bowel, and 2 kidney specific) were in the 5% of markers with the highest fold change. Conclusions We showed that expression analysis carried out with TLDA and qNPA are comparable. The qNPA technology developed by HTG Molecular Diagnostics, Inc. is a good alternative to assess expression in biopsies. We found that the HTGIOE could be an excellent automated tool to evaluate panels with low number of markers in a clinical lab. The HTGESOB allows evaluation of a large number of genes, but it includes a NGS step that makes it costlier and time consuming. It is a good exploratory tool for searching new markers.

Matrix dimensionality and stiffness cooperatively regulate osteogenesis of mesenchymal stromal cells

Osteogenic potential of mesenchymal stromal cells (MSCs) is mechanosensitive. It’s affected by the mechanical properties of the cellular microenvironment, particularly its mechanical modulus. To explore the effect of mechanical modulus on osteogenesis in the third dimension (3D), this study used a novel polyacrylamide (PA) scaffold whose pores are monodisperse and spherical, the mechanical moduli of which can be tuned across a wide range. It was found that MSCs have similar proliferation rates in PA scaffolds independent of the matrix stiffness. The contractile force exerted by MSCs inside PA scaffolds was strong enough to deform the pores of scaffolds made of more compliant PAs (whose shear modulus, Gscaffold′<4kPa). Only scaffolds of the highest stiffness (Gscaffold′=12kPa) can withhold the contraction from MSCs. After osteogenic induction for 21days, the expression profiles of marker genes showed that PA scaffolds of Gscaffold′=12kPa promoted osteogenesis of MSCs. Confocal image analysis demonstrated that there are more F-actin cytoskeletons and bundled stress fibers at higher matrix moduli in 2D and 3D. Moreover, the 3D porous structure promotes osteogenesis of MSCs more than 2D flat substrates. Together, the differences of cellular behaviors when cultured in 2D and 3D systems are evident. The PA scaffolds developed in the present study can be used for further investigation into the mechanism of MSC mechanosensing in the 3D context. Statement of SignificanceMechanical properties of the microenvironment affect cellular behaviors, such as matrix stiffness. Traditionally, cell biological investigations have mostly employed cells growing on 2D substrates. The 3D porous PA scaffolds with the same topological conformation and pore sizes but different stiffness generated in this study showed that the differences of cellular behaviors in 2D and 3D systems are evident. Our 3D scaffolds provide insights into tissue engineering when stem cells incorporated with 3D scaffolds and support the future studies of cellular mechanobiology as well as the elucidation the role mechanical factor plays on the physiology and fate determination of MSCs in the 3D context.

Transcription Profiles of Marker Genes Predict The Transdifferentiation Relationship between Eight Types of Liver Cell during Rat Liver Regeneration.

To investigate the transdifferentiation relationship between eight types of liver cell during rat liver regeneration (LR). 114 healthy Sprague-Dawley (SD) rats were used in this experimental study. Eight types of liver cell were isolated and purified with percoll density gradient centrifugation and immunomagentic bead methods. Marker genes for eight types of cell were obtained by retrieving the relevant references and databases. Expression changes of markers for each cell of the eight cell types were measured using microarray. The relationships between the expression profiles of marker genes and transdifferentiation among liver cells were analyzed using bioinformatics. Liver cell transdifferentiation was predicted by comparing expression profiles of marker genes in different liver cells. During LR hepatocytes (HCs) not only express hepatic oval cells (HOC) markers (including PROM1, KRT14 and LY6E), but also express biliary epithelial cell (BEC) markers (including KRT7 and KRT19); BECs express both HOC markers (including GABRP, PCNA and THY1) and HC markers such as CPS1, TAT, KRT8 and KRT18; both HC markers (KRT18, KRT8 and WT1) and BEC markers (KRT7 and KRT19) were detected in HOCs. Additionally, some HC markers were also significantly upregulated in hepatic stellate cells ( HSCs), sinusoidal endothelial cells (SECs) , Kupffer cells (KCs) and dendritic cells (DCs), mainly at 6-72 hours post partial hepatectomy (PH). Our findings indicate that there is a mutual transdifferentiation relationship between HC, BEC and HOC during LR, and a tendency for HSCs, SECs, KCs and DCs to transdifferentiate into HCs.