Transcriptome Remodeling in Gradual Development of Inverse Resistance between Paclitaxel and Cisplatin in Ovarian Cancer Cells.

Resistance to anti-cancer drugs is the main challenge in oncology. In pre-clinical studies, established cancer cell lines are primary tools in deciphering molecular mechanisms of this phenomenon. In this study, we proposed a new, transcriptome-focused approach, utilizing a model of isogenic cancer cell lines with gradually changing resistance. We analyzed trends in gene expression in the aim to find out a scaffold of resistance development process. The ovarian cancer cell line A2780 was treated with stepwise increased concentrations of paclitaxel (PTX) to generate a series of drug resistant sublines. To monitor transcriptome changes we submitted them to mRNA-sequencing, followed by the identification of differentially expressed genes (DEGs), principal component analysis (PCA), and hierarchical clustering. Functional interactions of proteins, encoded by DEGs, were analyzed by building protein-protein interaction (PPI) networks. We obtained human ovarian cancer cell lines with gradually developed resistance to PTX and collateral sensitivity to cisplatin (CDDP) (inverse resistance). In their transcriptomes, we identified two groups of DEGs: (1) With fluctuations in expression in the course of resistance acquiring; and (2) with a consistently changed expression at each stage of resistance development, constituting a scaffold of the process. In the scaffold PPI network, the cell cycle regulator-polo-like kinase 2 (PLK2); proteins belonging to the tumor necrosis factor (TNF) ligand and receptor family, as well as to the ephrin receptor family were found, and moreover, proteins linked to osteo- and chondrogenesis and the nervous system development. Our cellular model of drug resistance allowed for keeping track of trends in gene expression and studying this phenomenon as a process of evolution, reflected by global transcriptome remodeling. This approach enabled us to explore novel candidate genes and surmise that abrogation of the osteomimic phenotype in ovarian cancer cells might occur during the development of inverse resistance between PTX and CDDP.

High-resolution allele frequencies for NGS based HLA-A, B, C, DQB1 and DRB1 typing of 23,595 bone marrow donors recruited for the Polish central potential unrelated bone marrow donor registry.

Next-generation sequencing (NGS)-based typings of HLA-A, B, C, DQB1 and DRB1 loci were performed from 2018 to 2019 in 23 595 newly recruited or re-typed adult potential bone marrow donors registered in Poltransplant Registry to characterize allele and haplotype frequencies of HLA system for loci important for hematopoietic stem cell transplantation. The donors were recruited for registry and not for any other purpose including controls in a disease association study. The population sample was collected in various regions of Poland including all voivodships. The data regarding the degree of relatedness among individuals in the sample were not collected. Typings were supported by public funds as a part of the Polish National Program for Transplant Medicine Development. HLA frequency data are available in the Allele Frequencies Net Database.

The Defense Response of Nicotiana benthamiana to Peanut Stunt Virus Infection in the Presence of Symptom Exacerbating Satellite RNA.

Peanut stunt virus (PSV) is a widespread disease infecting legumes. The PSV strains are classified into four subgroups and some are defined by the association of satellite RNAs (satRNAs). In the case of PSV, the presence of satRNAs alters the symptoms of disease in infected plants. In this study, we elucidated the plant response to PSV-G strain, which occurs in natural conditions without satRNA. However, it was found that it might easily acquire satRNA, which exacerbated pathogenesis in Nicotiana benthamiana. To explain the mechanisms underlying PSV infection and symptoms exacerbation caused by satRNA, we carried out transcriptome profiling of N. benthamiana challenged by PSV-G and satRNA using species-specific microarrays. Co-infection of plants with PSV-G + satRNA increased the number of identified differentially expressed genes (DEGs) compared with the number identified in PSV-G-infected plants. In both treatments, the majority of up-regulated DEGs were engaged in translation, ribosome biogenesis, RNA metabolism, and response to stimuli, while the down-regulated DEGs were required for photosynthesis. The presence of satRNA in PSV-G-infected plants caused different trends in expression of DEGs associated with phosphorylation, ATP binding, and plasma membrane.

Gene expression profiling of acute myeloid leukemia samples from adult patients with AML-M1 and -M2 through boutique microarrays, real-time PCR and droplet digital PCR.

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American­British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1 gene expression levels correlated with the response to therapy. CAT expression was highest in patients who remained longer under complete remission, whereas WT1 expression increased with treatment resistance. On the whole, this study demonstrates that the AML-array can potentially serve as a first-line screening tool, and may be helpful for the diagnosis of AML, whereas the differentiation between AML subgroups can be more successfully performed with PCR-based analysis of a few marker genes.

A gene expression microarray for Nicotiana benthamiana based on de novo transcriptome sequence assembly.

BACKGROUND: Nicotiana benthamiana has been widely used in laboratories around the world for studying plant-pathogen interactions and posttranscriptional gene expression silencing. Yet the exploration of its transcriptome has lagged behind due to the lack of both adequate sequence information and genome-wide analysis tools, such as DNA microarrays. Despite the increasing use of high-throughput sequencing technologies, the DNA microarrays still remain a popular gene expression tool, because they are cheaper and less demanding regarding bioinformatics skills and computational effort. RESULTS: We designed a gene expression microarray with 103,747 60-mer probes, based on two recently published versions of N. benthamiana transcriptome (v.3 and v.5). Both versions were reconstructed from RNA-Seq data of non-strand-specific pooled-tissue libraries, so we defined the sense strand of the contigs prior to designing the probe. To accomplish this, we combined a homology search against Arabidopsis thaliana proteins and hybridization to a test 244k microarray containing pairs of probes, which represented individual contigs. We identified the sense strand in 106,684 transcriptome contigs and used this information to design an Nb-105k microarray on an Agilent eArray platform. Following hybridization of RNA samples from N. benthamiana roots and leaves we demonstrated that the new microarray had high specificity and sensitivity for detection of differentially expressed transcripts. We also showed that the data generated with the Nb-105k microarray may be used to identify incorrectly assembled contigs in the v.5 transcriptome, by detecting inconsistency in the gene expression profiles, which is indicated using multiple microarray probes that match the same v.5 primary transcripts. CONCLUSIONS: We provided a complete design of an oligonucleotide microarray that may be applied to the research of N. benthamiana transcriptome. This, in turn, will allow the N. benthamiana research community to take full advantage of microarray capabilities for studying gene expression in this plant. Additionally, by defining the sense orientation of over 106,000 contigs, we substantially improved the functional information on the N. benthamiana transcriptome. The simple hybridization-based approach for detecting the sense orientation of computationally assembled sequences can be used for updating the transcriptomes of other non-model organisms, including cases where no significant homology to known proteins exists.

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification.

The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes.

Time course transcriptional profiling of senescing barley leaves.

Cell senescence occurs as a part of developmental or stress-induced process. It is tightly regulated and involves a sequence of metabolic and structural alterations, eventually leading to cell death. Dark-induced leaf senescence is a useful model for studying senescence-related events. To facilitate the integration of physiological and molecular studies utilizing this model, we generated the microarray data set providing time course gene expression profiles in senescing barley leaves. Here, we describe the detailed procedures and data analysis scheme of our experiment. The entire data set (available at NCBI/GEO database under GSE62539) has been successively explored to find the genes differentially expressed during the senescence process as well as to identify genes with the invariant expression as reliable references for qPCR or ddPCR experiments.

Selection of reference genes for qPCR- and ddPCR-based analyses of gene expression in Senescing Barley leaves.

Leaf senescence is a tightly regulated developmental or stress-induced process. It is accompanied by dramatic changes in cell metabolism and structure, eventually leading to the disintegration of chloroplasts, the breakdown of leaf proteins, internucleosomal fragmentation of nuclear DNA and ultimately cell death. In light of the global and intense reorganization of the senescing leaf transcriptome, measuring time-course gene expression patterns in this model is challenging due to the evident problems associated with selecting stable reference genes. We have used oligonucleotide microarray data to identify 181 genes with stable expression in the course of dark-induced senescence of barley leaf. From those genes, we selected 5 candidates and confirmed their invariant expression by both reverse transcription quantitative PCR and droplet digital PCR (ddPCR). We used the selected reference genes to normalize the level of the expression of the following senescence-responsive genes in ddPCR assays: SAG12, ICL, AGXT, CS and RbcS. We were thereby able to achieve a substantial reduction in the data variability. Although the use of reference genes is not considered mandatory in ddPCR assays, our results show that it is advisable in special cases, specifically those that involve the following conditions: i) a low number of repeats, ii) the detection of low-fold changes in gene expression or iii) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.

VEGF is important for early liver regeneration after partial hepatectomy.

BACKGROUND: The aim of the study was to determine the role of Vascular Endothelial Growth Factor (VEGF) on the microvasculature and on angiogenetic gene expression after partial hepatectomy (PH) in the rat model. METHODS: To determine the effect of exogenous and endogenous VEGF after PH, rats were subjected to 70% PH and treated either with VEGF, anti-VEGF or NaCl. Postoperatively (3-168 h), vessel density (VD), vessel diameter (VDi), and intersinusoidal space, liver body weight ratio (LBR), hepatic proliferation and biochemical markers were assessed. To further elucidate the underlying molecular mechanisms hepatic gene expression was determined by customized cDNA arrays and quantitative RT-PCR. RESULTS: In the VEGF group, VD, VDi, and LBR were significantly increased compared with anti-VEGF or controls. Blockage of endogenous VEGF led to a marked increase of biochemical markers. Anti-VEGF almost completely suppressed and VEGF markedly enhanced hepatic proliferation in the first 24 h after surgery. This was associated with a modulation of cell cycle control genes (PC4, Gadd45a, Tis21/BTG2), v-jun, and CD14 by VEGF. CONCLUSIONS: VEGF plays an important role in liver regeneration and this may be due in part through its effects on neovascularization. Whether it may, when given therapeutically, represent a strategy to optimize liver regeneration in problematic patients needs to be clarified.

[Severe acute respiratory syndrome (SARS)--present status]. / Ciezki ostry zespól oddechowy (SARS)--stan obecny.

Basic facts about SARS are presented, including epidemiology, clinical symptoms and the course of disease, multinational search of etiological factor, diagnostic approaches and others. The role of molecular biology in elucidating structure, features and phylogenetic aspects of SARS coronavirus is underlined.