Digital Gene Expression Profiling Research Articles

Comparative Transcriptome Analysis Provides Insights into Response of Ulva compressa to Fluctuating Salinity Conditions.

Ulva compressa, a green tide-forming species, can adapt to hypo-salinity conditions, such as estuaries and brackish lakes. To understand the underlying molecular mechanisms of hypo-salinity stress tolerance, transcriptome-wide gene expression profiles in U. compressa were created using digital gene expression profiles. The RNA-seq data were analyzed based on the comparison of differently expressed genes involved in specific pathways under hypo-salinity and recovery conditions. The up-regulation of genes in photosynthesis and glycolysis pathways may contribute to the recovery of photosynthesis and energy metabolism, which could provide sufficient energy for the tolerance under long-term hyposaline stress. Multiple strategies, such as ion transportation and osmolytes metabolism, were performed to maintain the osmotic homeostasis. Additionally, several long noncoding RNA were differently expressed during the stress, which could play important roles in the osmotolerance. Our work will serve as an essential foundation for the understanding of the tolerance mechanism of U. compressa under the fluctuating salinity conditions.

Gene expression profiling reveals candidate genes for defining spider silk gland types

Molecular studies of the secretory glands involved in spider silk production have revealed candidate genes for silk synthesis and a complicated history of spider silk gene evolution. However, differential gene expression profiles of the multiple silk gland types within an individual orb-web weaving spider are lacking. Each of these gland types produces a functionally distinct silk type. Comparison of gene expression among spider silk gland types would provide insight into the genes that define silk glands generally from non-silk gland tissues, and the genes that define silk glands from each other. Here, we perform 3’ tag digital gene expression profiling of the seven silk gland types of the silver garden orb weaver Argiope argentata. Five of these gland types produce silks that are non-adhesive fibers, one silk includes both fibers and glue-like adhesives, and one silk is exclusively glue-like. We identify 1275 highly expressed, significantly upregulated, and tissue specific silk gland specific transcripts (SSTs). These SSTs include seven types of spider silk protein encoding genes known as spidroin genes. We find that the fiber-producing major ampullate and minor ampullate silk glands have more similar expression profiles than any other pair of glands. We also find that a subset of the SSTs is enriched for transmembrane transport and oxidoreductases, and that these transcripts highlight differences and similarities among the major ampullate, minor ampullate, and aggregate silk glands. Furthermore, we show that the wet glue-producing aggregate glands have the most unique SSTs, but still share some SSTs with fiber producing glands. Aciniform glands were the only gland type to share a majority of SSTs with other silk gland types, supporting previous hypotheses that duplication of aciniform glands and subsequent divergence of the duplicates gave rise to the multiple silk gland types within an individual spider.

Use of Single Cell RNA seq to Discern Molecular Pathways of Fibrosis and Predicting Emergence of CLAD

<h3>Purpose</h3> CLAD occurs after airway epithelial cell damage in transplant. The mechanisms of fibrosis are poorly understood. Information of which genes are involved at a single-cell level is crucial. Challenges studying distal airway: 1) Microarray gene expression analysis is limited to pooling all cells in the sample, not single cells, adding uncertainty; and 2) Flow cytometry cannot identify specific epithelial cells. BAL has airway epithelial cells (AEC) and immune cells. The purposes of this pilot study are: 1) determine if AEC can be successfully analyzed with the single cell transcriptomic analysis on clinical BAL samples, 2) what cell populations are analyzed, 3) can rare cells be isolated? <h3>Methods</h3> 5 ml BAL samples were aliquoted from routine clinically scheduled graft monitoring procedures in 3 patients. Cells were washed in PBS, then plated onto array of picoliter wells. Beads bearing cell- and transcript-barcoding capture probes is added along single cell. Each mRNA within a cell is specifically labeled. cells lysed, mRNAs hybridize to beads, reverse transcribed, amplified, and sequenced. <h3>Results</h3> The digital gene expression profile for each cell is assigned to the cell of origin and quantified. Rare AECs were isolated, mphages abundant. Expression patterns from individual cells were quantified with DESeq to identify significant differences in molecular count data. DESeq groups data points that are similar (gene expression type and number of transcripts per gene) in the raw data, then presents them closely approximated to one another in 2D data space. Each cluster represents a cell type. Each point a single cell. Cells with similar HLA expression profiles are the same color. Figure 1 presents the results in a tSNE plot. The tables indicate gene expression data at single cell level. <h3>Conclusion</h3> In this pilot study, we discovered avenues of analysis applicable to clinical translational studies. Next-gen techniques are essential to gain the knowledge needed to counter CLAD and foster new therapeutics.

Inter- and intra-tumor heterogeneity of metastatic prostate cancer determined by digital spatial gene expression profiling

Metastatic prostate cancer (mPC) comprises a spectrum of diverse phenotypes. However, the extent of inter- and intra-tumor heterogeneity is not established. Here we use digital spatial profiling (DSP) technology to quantitate transcript and protein abundance in spatially-distinct regions of mPCs. By assessing multiple discrete areas across multiple metastases, we find a high level of intra-patient homogeneity with respect to tumor phenotype. However, there are notable exceptions including tumors comprised of regions with high and low androgen receptor (AR) and neuroendocrine activity. While the vast majority of metastases examined are devoid of significant inflammatory infiltrates and lack PD1, PD-L1 and CTLA4, the B7-H3/CD276 immune checkpoint protein is highly expressed, particularly in mPCs with high AR activity. Our results demonstrate the utility of DSP for accurately classifying tumor phenotype, assessing tumor heterogeneity, and identifying aspects of tumor biology involving the immunological composition of metastases.

The effect of heat stress on proliferation, synthesis of steroids, and gene expression of duck granulosa cells.

Granulosa cells (GCs) play an important role in the development of follicles. In this study, we investigate the impact of heat stress at 41°C and 43°C on duck GCs' proliferation and steroids secretion. And, the transcriptomic responses to heat treatment were examined using RNA-sequencing analysis. Digital gene expression profiling was used to screen and identify differentially expressed genes (fold change ≥ 2 and Q value < 0.05). Further, the differential expression genes (DEGs) were classified into GO categories and KEGG pathways. The results show that duck GCs blocked in the G1 phase were increased on exposure to heat stress. Meanwhile, the expression of proliferative genes, which were essential for the transition from G1 to S phase, was inhibited. At the same time, heat stress inhibited the estradiol synthesis of GCs by decreasing CYP11A1 and CYP19A1 gene expression. A total of 241 DEGs including 181 upregulated and 60 downregulated ones were identified. Transcriptome result shows that heat shock protein and CXC chemokines gene were significantly activated during heat stress. While collagenases (MMP1 and MMP13) and strome lysins (MMP3) were downregulated. And, the hedgehog signaling pathway may be a prosurvival adaptive response under heat stress. These results offer a basis for better understanding the molecular mechanism underlying lay-eggs-less in ducks under heat stress.

Digital Gene Expression Analysis of Huanglongbing Affected Mandarins (Citrus reticulata Blanco) In Response to Thermotherapy

Huanglongbing (HLB), which is mainly associated with “Candidatus Liberibacter asiaticus” (CLas), is currently threatening citrus production around the world. This destructive century-old disease results in callose deposition in phloem plasmodesmata and starch overaccumulation in leaf chloroplasts. Heat treatment with high humidity has proven to be effective in eliminating the CLas titers in potted citrus trees. This study explored the curative mechanisms and impact of heat treatment (from 32 to 48 °C with 75% ± 5% relative humidity, 14 h in light and 10 h in dark for four days) on mandarin trees (Citrus reticulata Blanco) that were severely infected by CLas using Solexa/Illumina's digital gene expression (DGE) profiling. A total of 838 differentially expressed genes (DEGs) were identified, of which 510 were downregulated. Protein-protein interaction analysis of the DEGs indicated that the thermotherapy of HLB trees downregulated the circadian clock related genes in chloroplasts and upregulated the RNA-editing process in the mitochondria, characterized by 141 genes encoding pentatricopeptide repeat-containing proteins (PPR) being all upregulated. Furthermore, the expression of phenylpropanoid and flavonoid biosynthesis genes, and auxin-induced genes were suppressed. The defense response pathway of the thermotherapy treated diseased trees was apparently disrupted, as indicated by the downregulation of LRR receptor-like serine/threonine-protein kinase FLS2 gene. This study improves our understanding of the curative mechanisms of heat treatment on HLB trees and CLas biological regulation. Importantly, this provides new insights on the involvement of RNA modification by PPR genes and changes in the circadian clock after the heat stress treatment of CLas-infected citrus.

Digital gene expression profiling analysis of A549 cells cultured with PM10 in moxa smoke

Moxibustion is a traditional Chinese medicine therapy to cure diseases by fumigating meridians or affected parts via burning of moxa floss. Moxa smoke (MS) is one of the key factors in moxibustion. In this study, we adopted digital gene expression profiling, a next-generation gene sequencing technology, to investigate the effect of MS, inhalable particulate matter (PM 10 ), on human lung adenocarcinoma A549 cells. The effects of MS PM 10 on A549 cells, over different treatment durations were investigated in different groups: the 4-h group (4-h MS group and 4-h control group) and the 20-h group (20-h MS group and 20-h control group). Samples collected from the four groups were stored at −80 °C for subsequent digital gene expression analysis. The differentially expressed genes (DEGs), identified after PM 10 treatment, were screened, and their expression patterns analyzed by cluster analysis, Gene Ontology term enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with two control groups, 1109 DEGs were identified after 4 h of MS intervention and 3565 DEGs were found after 20 h of MS intervention, respectively. Compared with that after 4-h intervention, 2149 DEGs were identified after 20-h intervention. Cluster analysis demonstrated that PM 10 can significantly inhibit cell cycle process with the prolongation of intervention time. Significant pathway enrichment analysis showed that MS PM 10 can inhibit A549 cell cycle process at all phases. When MS PM 10 exposure time prolongs, the inhibitory effect on cell cycle process becomes more obvious. MS PM 10 has many biological activities, and may cause differential expression of genes involved in various biological processes. Nevertheless, further research on MS is warranted for better understanding of the mechanistic details.

Clinical laboratory validation of the MCL35 assay for molecular risk stratification of mantle cell lymphoma

Mantle cell lymphoma (MCL) is a clinically heterogeneous B cell malignancy for which a variety of prognostic factors have been proposed. Previously, a digital gene expression profiling “proliferation signature” capable of risk stratifying MCL was identified and subsequently developed into a multi-analyte prognostic assay, known as the “MCL35” assay. In this study, we sought to explore the performance characteristics of the MCL35 assay in a clinical laboratory and compare results with the Ki67 proliferation marker. The results describe the clinical validation of the MCL35 assay for molecular risk stratification of MCL including accuracy, sensitivity, specificity, use in acid-decalcified bone marrow core biopsies, fixatives, lower limit of RNA input, quality metrics, and other laboratory parameters. The resulting data indicate that this is a robust technique with outstanding reproducibility. Overall, the data support the concept of molecular signatures, as assessed with digital gene expression profiling, for improved standardization and reproducibility for proliferation assessment in MCL.

Characterization of the DREBA4-Type Transcription Factor (SlDREBA4), Which Contributes to Heat Tolerance in Tomatoes.

Dehydration-responsive element binding (DREB) transcription factors play crucial regulatory roles in abiotic stress. The only DREB transcription factor in tomato (Solanum lycopersicum), SlDREBA4 (Accession No. MN197531), which was determined to be a DREBA4 subfamily member, was isolated from cv. Microtom using high-temperature-induced digital gene expression (DGE) profiling technology. The constitutive expression of SlDREBA4 was detected in different tissues of Microtom plants. In addition to responding to high temperature, SlDREBA4 was up-regulated after exposure to abscisic acid (ABA), cold, drought and high-salt conditions. Transgenic overexpression and silencing systems revealed that SlDREBA4 could alter the resistance of transgenic Microtom plants to heat stress by altering the content of osmolytes and stress hormones, and the activities of antioxidant enzymes at the physiologic level. Moreover, SlDREBA4 regulated the downstream gene expression of many heat shock proteins (Hsp), as well as calcium-binding protein enriched in the pathways of protein processing in endoplasmic reticulum (ko04141) and plant-pathogen interaction (ko04626) at the molecular level. SlDREBA4 also induces the expression of biosynthesis genes in jasmonic acid (JA), salicylic acid (SA), and ethylene (ETH), and specifically binds to the DRE elements (core sequence, A/GCCGAC) of the Hsp genes downstream from SlDREBA4. This study provides new genetic resources and rationales for tomato heat-tolerance breeding and the heat-related regulatory mechanisms of DREBs.

Glutamate-Cysteine Ligase Catalytic Subunit Attenuated Hepatitis C Virus-Related Liver Fibrosis and Suppressed Endoplasmic Reticulum Stress

The study aimed to clarify the role and molecular mechanism of glutamate-cysteine ligase catalytic subunit (GCLC) in modulating Hepatitis C virus (HCV)-related liver fibrosis. Twenty patients with HCV-related liver fibrosis and 15 healthy controls were enrolled. Differentially expressed plasma mRNAs were detected by digital gene expression profile analysis and validated by qRT-PCR. Hepatic histopathology was observed by H&E and Masson stained liver sections. The mRNA and protein expression of GCLC, endoplasmic reticulum (ER) stress markers, and inflammatory and fibrogenic factors were detected in liver tissues from patients with HCV-related hepatic fibrosis and HCV core protein-expressing LX-2. The GCLC-overexpressing LX-2 were established by transfecting puc19-GCLC plasmid. Then, glutathione and reactive oxygen species (ROS) levels were measured respectively by spectrophotometric diagnostic kit and dihydrodichlorofluorescein diacetate kit. GCLC were dramatically down-regulated in HCV-related fibrotic livers and activated HSCs, which companied with up-regulation of ER stress-related genes, including inositol-requiring 1 (IRE1) and glucose-regulated protein 78 (GRP78). Also, the proinflammatory and profibrogenic gene, including nuclear factor kappa B (NF-κB), tumor necrosis factor α (TNFα), and transforming growth factor 1(TGFβ1), was highly upregulated. Overexpression of GCLC in hepatic stellate cells could suppress α-SMA and collagen I expression, produce hepatic GSH and reduce ROS, and down-regulate IRE1, GRP78, NF-κB, TNF-α, and TGFβ1 expression. GCLC was a negative regulatory factor in the development of HCV-related liver fibrosis and might be a potential therapeutic target for liver fibrosis.

Transcriptional profile of ovine oocytes matured under lipopolysaccharide treatment in vitro

Lipopolysaccharide (LPS) derived from gram negative bacteria cell wall is known to cause ruminal acidosis and/or infectious diseases such as metritis and mastitis which has a significant negative impact on the reproductive performance. This study aimed to investigate the effect of LPS on oocyte maturation and subsequent development in vitro. Ovine cumulus oocyte complexes (COCs) were matured in a medium supplemented with 0 (control), 0.01, 0.1, 1 and 10 μg/mL LPS. Nuclear maturation, cleavage and blastocyst rate, mitochondrial membrane potential (ΔΨm), intracellular reactive oxygen species (ROS) content and changes to the transcript abundance were evaluated. In case of the maturation rate, the percentage of oocytes reaching the MII stage was lower following exposure to 10 μg/mL LPS in comparison to the control group (P < 0.05). Moreover, the blastocyst rate decreased in case of 1 and 10 μg/mL LPS when compared to the control group (P < 0.05). ROS overproduction accompanied by a decreased ΔΨm were recorded in LPS treated oocytes in comparison to the control group (P < 0.05). The 3′ tag digital gene expression profiling method revealed that 7887 genes were expressed while only seven genes exhibited changes in the transcript abundance following exposure to LPS. Tripartite motif containing 25 (TRIM25), Tripartite motif containing 26 (TRIM26), Zona Pellucida glycoprotein 3 (ZP3), Family with sequence similarity 50-member A (FAM50A), Glyoxalate and hydroxy pyruvate reductase (GRHPR), NADH ubiquinase oxireductase subunit A8 (NDUFA8) were down-regulated (P < 0.05), while only Centrin 3 (CETN3) was up-regulated (P < 0.05). Our findings show that LPS has undesirable effects on the maturation competence of ovine oocytes and subsequent embryo development. In addition, the transcriptomic profiling results may shed more light on the molecular mechanisms of LPS-induced infertility in ruminants.

Predation threats for a 24-h period activated the extension of axons in the brains of Xenopus tadpoles

The threat of predation is a driving force in the evolution of animals. We have previously reported that Xenopus laevis enhanced their tail muscles and increased their swimming speeds in the presence of Japanese larval salamander predators. Herein, we investigated the induced gene expression changes in the brains of tadpoles under the threat of predation using 3′-tag digital gene expression profiling. We found that many muscle genes were expressed after 24 h of exposure to predation. Ingenuity pathway analysis further showed that after 24 h of a predation threat, various signal transduction genes were stimulated, such as those affecting the actin cytoskeleton and CREB pathways, and that these might increase microtubule dynamics, axonogenesis, cognition, and memory. To verify the increase in microtubule dynamics, DiI was inserted through the tadpole nostrils. Extension of the axons was clearly observed from the nostril to the diencephalon and was significantly increased (P ≤ 0.0001) after 24 h of exposure to predation, compared with that of the control. The dynamic changes in the signal transductions appeared to bring about new connections in the neural networks, as suggested by the microtubule dynamics. These connections may result in improved memory and cognition abilities, and subsequently increase survivability.

Clusterin contributes to hepatitis C virus-related hepatocellular carcinoma by regulating autophagy

AimsTo explore the potential regulatory mechanism of differentially expressed mRNAs in Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Main methodsPatients with HCV-related HCC and age- and gender-matched healthy subjects were enrolled. Differentially expressed mRNAs in the plasma were detected by digital gene expression (DGE) profile analysis. HepG2 and SMMC7721 cells stably transfected with HCV-core protein and the control plasmid were established. And small interfering RNA (siRNA) was used to knockdown the target gene in HCV core-expressing HCC cell lines. mRNA expression was determined by qRT-PCR. Protein expression was measured by Western blot and immunohistochemistry staining. Key findingsDGE profile data showed aberrant mRNA expression contributed to the progression of HCV-HCC, and clusterin (CLU), which was significantly highly expressed, was chosen as a candidate gene. Further evidence showed CLU was highly expressed in tumor tissues of HCV-HCC patients and HCV core-expressing HCC cell lines, accompanied with enhanced autophagy and upregulation of pro-autophagy genes. And knockdown of CLU in HCC cell lines suppressed cell autophagy, which was indicated by decreased expression of autophagy marker light chain 3B (LC3B) ІІ/І ratio, and downregulated pro-autophagy genes like Beclin1, autophagy-related protein 7 (Atg7) and Lamp2. On the other hand, anti-autophagy genes or regulators, including p62 and phosphorylated mammalian target of rapamycin (p-mTOR), were notably upregulated. SignificanceCLU could promote the progression of HCV-related HCC by regulating autophagy, which might be a potential therapeutic target of HCV-HCC.

Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system

BackgroundDigital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System’s ability to reproducibly quantify the expression of 730 immune genes from skin biopsies.MethodsHealthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at − 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at − 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at − 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes.ResultsExcept for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols.ConclusionsA variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.

Protein- and sequencing-based massively parallel single-cell approaches to immune profiling asthma-specific cell subtypes and related gene expression biomarkers

Abstract We implemented the BD Rhapsody Single-Cell Analysis System to assess cellular heterogeneity present in twins by capturing and sequencing over 40000 single cells across 10 samples from 5 twins patients with asthma or without asthma using oligos-conjugated antibodies and the whole blood immune targeted gene panel that utilizes multiplex PCR for detection of over 500 genes for immune cells subtyping, immunophenotyping, cell-type identification and markers correlated with stimulations. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We demonstrate the ability of the BD Rhapsody targeted panel to distinguish six different cell types and 18 distinct gene-expression biomarkers, including CLEC4E, CSF2, IL1RN, TPSAB1, CCL19, CCL22, CXCL2, LAMP3, SPP1, and VMO etc.. The HLA-DRA, HLA-DQB1, SPP1, and IL1RN had allelic polymorphism related to asthma. The BD Rhapsody targeted panel yields more sequencing depth with much fewer (&amp;lt;2%) sequencing reads per cell. The measurement of specific proteins and transcripts in individual cells is critical for understanding the role of cellular diversity in development, health, and disease.

Low-Copy Genes in Terpenoid Metabolism: The Evolution and Expression of MVK and DXR Genes in Angiosperms.

Terpenoids are a diverse class of metabolites that impact plant metabolism in response to environmental cues. They are synthesized either via a predominantly cytosolic (MVA) pathway or a plastidic pathway (MEP). In Arabidopsis, several enzymes from the MVA and MEP pathways are encoded by gene families, excluding MVK and DXR, which are single-copy genes. In this study, we assess the diversity, evolution and expression of DXR and MVK genes in selected angiosperms and Coffea arabica in particular. Evolutionary analysis revealed that DXR and MVK underwent purifying selection, but the selection effect for DXR was stronger than it was for MVK. Digital gene expression (DGE) profile analysis of six species revealed that expression levels of MVK in flowers and roots were high, whereas for DXR peak values were observed in leaves. In C. arabica, both genes were highly expressed in flowers, and CaDXR was upregulated in response to methyl jasmonate. C. arabica DGE data were validated by assessing gene expression in selected organs, and by plants treated with hexanoic acid (Hx) using RT-qPCR. MVK expression was upregulated in roots treated with Hx. CaDXR was downregulated in leaves by Hx treatment in a genotype-specific manner, indicating a differential response to priming.

Use of Single Cell RNA Sequencing to Discern Molecular Pathways of Fibrosis and Predicting Emergence of CLAD

Chronic Lung Allograft Dysfunction (CLAD) is characterized by airway epithelial cell damage followed by airway and parenchymal fibrosis. The origin and initiation of fibrosis are notunderstood. To understand how cells differ from each other, we need to understand which genes are transcribed at a single-cell level. The purpose of pilot to: 1) determine if AECs can be analyzed with scRNAseq 2) what cell populations can we identify 3) can we find rare AEC pops METHODS: 5 ml BAL aliquots from surveillance procedures in 3 patients. Cells plated onto plates (10,000 wells). Beads with cell- and transcript-barcodes added alongside 1 cell to specifically label. cells lysed, mRNAs hybridized to beads, RT, amplified, and sequenced RESULTS: gene expression profile for each cell is reconstructed to the cell of origin. Figure presents the various cell types found in tSNE plot. Same cells close to one another in the 2D data space, each dot a cell. optimized to yield the highest number of AECs, but AM are present in abundance. Gene expression patterns were quantified. Each cluster a cell type. Cells with similar HLA expression profiles are the same color. rare cell types can be isolated and studied with scRNAseq. gene expression reveal T-cells in acute rejection, AECs involved in tissue remodeling and rare AEC progenitor cells (Table) implying EMT. This discovery pilot demonstrates the power to delve into cell-specific activity and can be further pursued to discover pathways that lead to CLAD long before graft dysfunction is measured.

ARF family identification in Tamarix chinensis reveals the salt responsive expression of TcARF6 targeted by miR167.

Auxin response factors (ARFs) are important transcription factors (TFs) that are differentially expressed in response to various abiotic stresses. The important roles of ARFs and small RNA-ARF pathways in mediating plant growth and stress responses have emerged in several recent studies. However, no studies on the involvement of ARFs in tamarisk trees, which are resistant to salinity, have been conducted. In this study, systematic analysis revealed 12 TcARF genes belonging to five different groups in Tamarix chinensis. The microRNA response elements of miR160, which belongs to group I and miR167, which belongs to group III, were conserved in terms of their location and sequence. Moreover, digital gene expression profiles suggested that a potential miR167 target gene, TcARF6, was rapidly expressed in response to salt stress. Cloning of TcARF6 revealed that TcARF6 could be an activation TF with a glutamine-rich region and expression pattern analysis revealed that the expression of TcARF6 was significantly downregulated specifically in the roots. A significant negative correlation in the expression pattern of tch-miR167/TcARF6 indicated that this module may play a key role in the response to salt stress. Overall, these results provide basic information on the posttranscriptional regulation of TcARF6 for future investigations of the T. chinensis salt-stress response.

The transmembrane protein MaSho1 negatively regulates conidial yield by shifting the conidiation pattern in Metarhizium acridum.

Sho1 is an important membrane sensor upstream of the HOG-MAPK signaling pathway, which plays critical roles in osmotic pressure response, growth, and virulence in fungi. Here, a Sho1 homolog (MaSho1), containing four transmembrane domains and one Src homology (SH3) domain, was characterized in Metarhizium acridum, a fungal pathogen of locusts. Targeted gene disruption of MaSho1 impaired cell wall integrity, virulence, and tolerances to UV-B and oxidative stresses, while none of them was affected when the SH3 domain was deleted. Intriguingly, disruption of MaSho1 significantly increased conidial yield, which was not affected in the SH3 domain mutant. Furthermore, it was found that deletion of MaSho1 led to microcycle conidiation of M. acridum on the normal conidiation medium. Deletion of MaSho1 significantly shortened the hyphal cells but had no effect on conidial germination. Digital gene expression profiling during conidiation indicated that differential expression of genes was associated with mycelial development, cell division, and differentiation between the wild type and the MaSho1 mutant. These data suggested that disruption of MaSho1 shifted the conidiation pattern by altering the transcription of genes to inhibit mycelial growth, thereby promoting the conidiation of M. acridum.

Transcriptome Profiling Reveals Candidate Genes Involved in Stem Swelling of Tumorous Stem Mustard

Tumorous stem mustard is well known for its swollen stem from which pickled “Fuling Mustard” is made. The molecular mechanisms governing the formation of the modified swollen stems are still poorly understood. This paper aims to identify candidate genes involved in the developmental regulation of the swollen stems. We sought to map previously published transcriptome datasets for Brassica juncea, including those derived from swollen stems at four different developmental stages and a mutant variety without swollen stems. Using pairwise comparisons of the five datasets, we identified 31 368 differentially expressed genes (DEGs). A total of 55 DEGs related to plant hormone signal transduction and 259 continuously up- or downregulated transcription factors were identified during stem development using Gene Ontology (GO) analysis. Quantitative real-time PCR (qRT-PCR) results showed that the expressions of 12 important candidate DEGs were consistent with RNA-seq results. Our study provided digital gene expression profiling and a dynamic view of the swollen stem development process. Furthermore, we identified candidate genes for further studies on mechanisms of modified stem development in non-model species.