Variation In Gene Expression Patterns Research Articles

Comparative Transcriptomic Analysis Reveals Domestication and Improvement Patterns of Broomcorn Millet (Panicum miliaceum L.).

Broomcorn millet (Panicum miliaceum L.) is one of the earliest crops, domesticated nearly 8000 years ago in northern China. It gradually spread across the entire Eurasian continent, as well as to America and Africa, with recent improvement in various reproductive and vegetative traits. To identify the genes that were selected during the domestication and improvement processes, we performed a comparative transcriptome analysis based on wild types, landraces, and improved cultivars of broomcorn millet at both seeding and filling stages. The variations in gene expression patterns between wild types and landraces and between landraces and improved cultivars were further evaluated to explore the molecular mechanisms underlying the domestication and improvement of broomcorn millet. A total of 2155 and 3033 candidate genes involved in domestication and a total of 84 and 180 candidate genes related to improvement were identified at seedling and filling stages of broomcorn millet, respectively. The annotation results suggested that the genes related to metabolites, stress resistance, and plant hormones were widely selected during both domestication and improvement processes, while some genes were exclusively selected in either domestication or improvement stages, with higher selection pressure detected in the domestication process. Furthermore, some domestication- and improvement-related genes involved in stress resistance either lost their functions or reduced their expression levels due to the trade-offs between stress resistance and productivity. This study provided novel genetic materials for further molecular breeding of broomcorn millet varieties with improved agronomic traits.

Genetic evolution of keratinocytes to cutaneous squamous cell carcinoma.

We performed multi-omic profiling of epidermal keratinocytes, precancerous actinic keratoses, and squamous cell carcinomas to understand the molecular transitions during skin carcinogenesis. Single-cell mutational analyses showed that most keratinocytes in normal skin had lower mutation burdens than melanocytes and fibroblasts, however keratinocytes with TP53 or NOTCH1 mutations had substantially higher mutation burdens, suggesting that these mutations prime keratinocytes for transformation by increasing their mutation rate. Mutational profiling and spatial transcriptomics on squamous cell carcinomas adjacent to actinic keratoses revealed TERT promoter and CDKN2A mutations emerging in actinic keratoses, whereas additional mutations inactivating ARID2 and activating the MAPK-pathway delineated the transition to squamous cell carcinomas. Spatial variation in gene expression patterns was common in both tumor and immune cells, with high expression of checkpoint molecules at the invasive front of tumors. In conclusion, this study documents key events during the evolution of cutaneous squamous cell carcinoma.

The pivotal role of phosphorus level gradient in regulating nitrogen cycle in wetland ecosystems

Phosphorus (P) is one of key drivers in Earth's nitrogen (N) cycle, however, the global overview of the P-regulated microbial community structure and gene abundance carrying wetland N process remains to be investigated. The key environmental factors that influenced wetland N cycle were initially screened, verifying the central role P. More complex and stable community interaction can be established in rich (20 mg/kg < P ≤ 100 mg/kg) and surplus P groups (P > 100 mg/kg) compared to that in deficient P group (P ≤ 20 mg/kg), with enhanced participation of betaproteobacteria and actinobacteria (i.e., changed hub microorganisms). Accordingly, P-mediated variations in gene expression patterns can be expected. On the one hand, the gene responses to carbon (C), N, and P factors presented nearly synchronous variation, highlighting the potential C-N-P coupling cycle in wetland ecosystem. On the other hand, the gene sensitivity towards environmental factors was changed at different P levels. Overall, the P level gradient can influence N cycle in direct (i.e., influences on gene abundances) and indirect (i.e., influences on gene response to environmental factors) manners. These findings provide important insights for controlling the N cycle in wetland ecosystems, particularly in cases where P levels are limiting factors.

Administration of Human-Derived Mesenchymal Stem Cells Activates Locally Stimulated Endogenous Neural Progenitors and Reduces Neurological Dysfunction in Mice after Ischemic Stroke.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin-GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP+ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry+ h-MSCs stimulated GFP+ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 104 cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

Abstract 5448: Malignant RNA editing by inflammation-responsive RNA editase ADAR1 drives T-cell leukemia relapse via attenuating dsRNA sensing

Abstract Leukemia-initiating cells (LICs) play a crucial role in leukemia research and treatment, being considered the source of relapse and resistance, posing a substantial challenge for successful management of the disease. It's crucial to identify the key factors that drive LIC self-renewal to develop targeted methods for eliminating LICs and preventing relapse. In this study, we demonstrate that the RNA editing enzyme ADAR1 plays a vital role as a determinant of stemness, promoting LIC self-renewal by reducing the detection of abnormal double-stranded RNA (dsRNA). As a result, ADAR1-knockdown significantly impairs the ability of LICs to self-renew and extends survival in T-ALL PDX models. We performed RNA-seq studies on enriched T-ALL LICs (CD34+Lin-). The lentivirus-to-cell ratio was optimized to achieve a 50% reduction in ADAR1, resulting in differential expression of 661 genes, with downregulation of critical self-renewal genes in ADAR1-deficient leukemia-initiating cells. Importantly, additional data indicated that ADAR1 likely influences self-renewal gene expression independently of A-to-I RNA editing, revealing novel regulatory mechanisms in leukemia. Knockdown of MDA5 and ADAR1 in patient-derived xenograft (PDX) T-ALL leukemia-initiating cells (LICs) revealed diverse rescue effects on self-renewal in different PDX models. The differential response was associated with variations in gene expression patterns in Rig-I-Like signaling and cytosolic sensing pathways, suggesting intrinsic differences in IFN signaling properties among patients. Moreover, the study proposes a patient-specific reliance on the ADAR1 p150-MDA5 axis for promoting self-renewal, as evidenced by differential rescue effects and varying expression levels of ADAR1 isoforms and MDA5 in T-ALL patient samples and cell lines. ADAR1, beyond its role in RNA editing, acts as a dsRNA binding protein, exerting RNA editing-independent functions to suppress aberrant IFN signaling and promote cancer progression. Using Jurkat cells with high intrinsic MDA5 levels, an ADAR1 knockout (KO) was generated, revealing that ADAR1 is upregulated by IFN treatment, predominantly the p150 isoform. We explored the dual roles of ADAR1—RNA editing-dependent and -independent—in suppressing IFN-induced apoptosis, demonstrating the necessity of both for optimal function. Additionally, ADAR1 was found to suppress cytoplasmic dsRNA sensing pathways, such as RIG-I and PKR, and its RNA editing-independent activity contributes significantly to the suppression of interferon-stimulated gene (ISG) expression in T-ALL. In summary, our findings reveal that ADAR1 acts as a factor that promotes self-renewal while limiting the recognition of naturally occurring dsRNA. Therefore, targeting ADAR1 emerges as a safe and effective therapeutic approach for eradicating LICs in T-ALL. Citation Format: Maria Dolores Rivera, Haoran Zhang, Qingchen Zhou, Jessica Pham, Jane Isquith, Roman Sasik, Adam Mark, Sabina Enlund, Frida Holm, Ma Wenxue, Kathleen Fisch, Dennis Kuo, Catriona Jamieson, Qingfei Jiang. Malignant RNA editing by inflammation-responsive RNA editase ADAR1 drives T-cell leukemia relapse via attenuating dsRNA sensing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5448.

The significance of single-cell transcriptome analysis in epididymis research.

As a crucial component of the male reproductive system, the epididymis plays multiple roles, including sperm storage and secretion of nutritive fluids for sperm development and maturation. The acquisition of fertilization capacity by sperm occurs during their transport through the epididymis. Compared with the testis, little has been realized about the importance of the epididymis. However, with the development of molecular biology and single-cell sequencing technology, the importance of the epididymis for male fertility should be reconsidered. Recent studies have revealed that different regions of the epididymis exhibit distinct functions and cell type compositions, which are likely determined by variations in gene expression patterns. In this research, we primarily focused on elucidating the cellular composition and region-specific gene expression patterns within different segments of the epididymis and provided detailed insights into epididymal function in male fertility.

Deciphering Glioblastoma: Fundamental and Novel Insights into the Biology and Therapeutic Strategies of Gliomas.

Gliomas constitute a diverse and complex array of tumors within the central nervous system (CNS), characterized by a wide range of prognostic outcomes and responses to therapeutic interventions. This literature review endeavors to conduct a thorough investigation of gliomas, with a particular emphasis on glioblastoma (GBM), beginning with their classification and epidemiological characteristics, evaluating their relative importance within the CNS tumor spectrum. We examine the immunological context of gliomas, unveiling the intricate immune environment and its ramifications for disease progression and therapeutic strategies. Moreover, we accentuate critical developments in understanding tumor behavior, focusing on recent research breakthroughs in treatment responses and the elucidation of cellular signaling pathways. Analyzing the most novel transcriptomic studies, we investigate the variations in gene expression patterns in glioma cells, assessing the prognostic and therapeutic implications of these genetic alterations. Furthermore, the role of epigenetic modifications in the pathogenesis of gliomas is underscored, suggesting that such changes are fundamental to tumor evolution and possible therapeutic advancements. In the end, this comparative oncological analysis situates GBM within the wider context of neoplasms, delineating both distinct and shared characteristics with other types of tumors.

Gene expression profiling on CML patients with Philadelphia translocation.

The use of tyrosine kinase inhibitors (TKIs) and other targeted therapeutics plays a pivotal role in treatment management for individuals diagnosed with chronic myeloid leukemia (CML). However, some patients may experience fewer favorable outcomes and treatment resistance. Our work aims to use whole transcriptome sequencing to evaluate the variations in gene expression patterns among individuals with CML based on their response to TKI therapy. Ten blood samples were obtained from two groups of patients diagnosed with CML: those at the initial diagnosis stage and those at the recurrence stage. RNA extraction was performed on all samples and used for next-generation sequencing. The data analysis was performed using the DESeq2 R program. In total, 499 genes were identified as having statistically significant differences in expression levels between the two groups. Of these, 122 genes exhibited upregulation, and 377 genes exhibited downregulation. We observed a notable dysregulation in the expression levels of NTRK2 (with a fold change more significant than +5). A significant proportion of the genes that were expressed demonstrated involvement in several biological processes, including the cell cycle, PI3K-AKT signaling system, cellular senescence, oxidative phosphorylation, microRNA in cancer, FOXO signaling pathway, P53 signaling pathway, and other related pathways. The results demonstrate a correlation between signaling pathways and the development of treatment resistance in patients with CML. These pathways exhibited enhanced efficacy in transmitting signals downstream of the TKI target, BCR-ABL. Several target genes require additional validation in a more extensive cohort study to verify their correlation with TKI resistance. The present research highlights that many BCR-ABL-independent pathways may be correlated with resistance, thus enhancing the prospective therapy options for patients with CML.

PROST: quantitative identification of spatially variable genes and domain detection in spatial transcriptomics

Computational methods have been proposed to leverage spatially resolved transcriptomic data, pinpointing genes with spatial expression patterns and delineating tissue domains. However, existing approaches fall short in uniformly quantifying spatially variable genes (SVGs). Moreover, from a methodological viewpoint, while SVGs are naturally associated with depicting spatial domains, they are technically dissociated in most methods. Here, we present a framework (PROST) for the quantitative recognition of spatial transcriptomic patterns, consisting of (i) quantitatively characterizing spatial variations in gene expression patterns through the PROST Index; and (ii) unsupervised clustering of spatial domains via a self-attention mechanism. We demonstrate that PROST performs superior SVG identification and domain segmentation with various spatial resolutions, from multicellular to cellular levels. Importantly, PROST Index can be applied to prioritize spatial expression variations, facilitating the exploration of biological insights. Together, our study provides a flexible and robust framework for analyzing diverse spatial transcriptomic data.

Blockade of histone deacetylase activity affects transcription and splicing of neuronal and glial genes

The study of the molecular mechanisms underlying plastic processes in the nervous system is of great interest in modern neuroscience. It is important to understand that epigenetic modifications, which are crucial for the development and cellular differentiation, can also be involved in plastic processes in the adult nervous system. In our early work, we provided evidence that the expression of important memory-related genes, such as Prkcz and Prkci, can be regulated epigenetically [1]. In the current study, we extended the previous work to the systemic level by applying the RNA sequencing approach to evaluate global changes in the expression patterns of various genes during the induction of epigenetic rearrangements. Rat cortical neuron cultures were incubated with one of the nonselective histone deacetylase inhibitors (trichostatin A, TSA; sodium butyrate, NaB) to change the level of epigenetic regulation. Next, the total RNA was extracted and subjected to RNA-Seq libraries preparation and subsequent NGS-sequencing. Bioinformatics analysis of transcriptomic data revealed substantial overlapping of differentially expressed genes (DEGs) in NaB-treated and TSA-treated groups, indicating that different histone deacetylase (HDAC) inhibitors induce transcriptional changes in primary neuron cultures through common regulatory pathways irrespective of chemical structure of applied inhibitor. We found that histone deacetylase blockade is accompanied by a transition from proliferative processes to cellular differentiation. Gene Ontology (GO) analysis of DEGs datasets revealed that the upregulated genes were engaged in cell differentiation and specialization, tissue and embryonic morphogenesis, and the development of various peripheral tissues and organs. On the contrary, genes that reduce the expression under induction of epigenetic rearrangements were involved in biological processes associated with cell proliferation and, most interestingly, the specialization of various brain cells (neurons, astrocytes, oligodendrocytes). It was shown that the expression of a number of glial markers typical for astrocytes and oligodendrocytes was significantly reduced after application of HDAC inhibitors, which was also confirmed by quantitative PCR using specific primer pairs on selected target genes. During the data analysis, we also found a significant decrease in the expression of various neuronal markers associated with the cytoskeleton, the organization of pre- and postsynaptic endings, synaptic transmission. It is known that fine-tuning of various processes in the central nervous system is due to the production of different isoforms of proteins from the same gene due to the process of alternative splicing of the resulting mRNA. According to the literature, epigenetic rearrangements create a certain environment for the regulation of alternative splicing of different genes [2, 3]. It is shown that production of alternative isoforms can play an important role in various plastic processes [4, 5]. Therefore, using IsoformSwitchAnalyzeR and DEXSeq packages we analyzed the possibility of alternative splicing during the induction of epigenetic rearrangements in rat cortical neuron cultures by evaluating the abundance of various transcripts based on exon usage. We found that some glial genes and a large number of neuronal genes, especially those associated with postsynaptic organization and cell communication, were alternatively spliced after application of histone deacetylase inhibitors. Inhibition of HDAC activity in cortical neuron cultures mainly affected the choice of alternative transcription starts (ATSS) and terminators (ATTS), and to a lesser extent alternative splicing of exons. Obtained results were selectively confirmed by the quantitative PCR using specific pairs of primers for individual exons of different transcript isoforms. Thus, within this study, it was found that histone deacetylases play an important role in the specialization of various brain cells, and the suppression of their activity affects the expression and alternative splicing of various glial and neuronal marker genes. We do not exclude that global transcriptome changes caused by alternative splicing will lead to qualitative rearrangements of the neuron network, and this is the direction of future research.

Molecular insights into the salt stress response of Pearl millet (Pennisetum glaucum): Pathways, differentially expressed genes and transcription factors

AbstractIntroductionPearl millet (Pennisetum glaucum) plays a crucial role as a cereal crop in arid and semi‐arid regions, where it confronts the formidable challenge of salt stress.Materials and MethodsTo unravel the underlying molecular mechanisms that underpin its salt stress resilience, we subjected 14‐day‐old seedlings to three distinct groups: Control, 75 mM NaCl and 150 mM NaCl. These pots received daily irrigation with their respective treatment solutions for a duration of 7 days. Following this week‐long treatment, we measured plant chlorophyll content, as well as the fresh and dry weights of shoots and roots. It became evident that the saline treatment, particularly in the 150 mM NaCl group, had a more pronounced impact on both weight and chlorophyll content in comparison to the control group, surpassing the effects observed in the 75 mM NaCl group. Subsequently, we conducted RNA sequence analysis on the leaves of Pearl millet from both the control and 150 mM NaCl‐treated groups.ResultsThe results revealed that 27.6% of Pennisetum glaucum genes exhibited differential expression, with 3246 genes being upregulated and 7408 genes downregulated when compared to the control group. Principal component analysis underscored distinct variations in gene expression patterns between the control and salt‐stressed groups. Pathway analysis sheds light on the upregulated differentially expressed genes (DEGs), highlighting their involvement in crucial pathways such as phytyl‐PP biosynthesis, lysine degradation, glutamate biosynthesis, nitrate assimilation and DLO biosynthesis. Conversely, the downregulated DEGs were associated with pathways like coumarins biosynthesis, pinobanksin biosynthesis, UDP‐ d‐glucuronate biosynthesis and cholesterol biosynthesis, among others. Furthermore, our transcription factor analysis unveiled specific families associated with the salt stress response, including bHLH, ERF, NAC, WRKY, bZIP, MYB and HD‐ZIP.ConclusionsThese findings represent a significant advancement in our comprehension of Pearl millet's capacity to withstand salt stress and provide potential targets for the development of salt‐resistant crops, contributing to the advancement of sustainable agriculture in regions affected by salinity.

Identification of Potential Sex-Specific Biomarkers in Pigs with Low and High Intramuscular Fat Content Using Integrated Bioinformatics and Machine Learning.

Intramuscular fat (IMF) content is a key determinant of pork quality. Controlling the genetic and physiological factors of IMF and the expression patterns of various genes is important for regulating the IMF content and improving meat quality in pig breeding. Growing evidence has suggested the role of genetic factors and breeds in IMF deposition; however, research on the sex factors of IMF deposition is still lacking. The present study aimed to identify potential sex-specific biomarkers strongly associated with IMF deposition in low- and high-IMF pig populations. The GSE144780 expression dataset of IMF deposition-related genes were obtained from the Gene Expression Omnibus. Initially, differentially expressed genes (DEGs) were detected in male and female low-IMF (162 DEGs, including 64 up- and 98 down-regulated genes) and high-IMF pigs (202 DEGs, including 147 up- and 55 down-regulated genes). Moreover, hub genes were screened via PPI network construction. Furthermore, hub genes were screened for potential sex-specific biomarkers using the least absolute shrinkage and selection operator machine learning algorithm, and sex-specific biomarkers in low-IMF (troponin I (TNNI1), myosin light chain 9(MYL9), and serpin family C member 1(SERPINC1)) and high-IMF pigs (CD4 molecule (CD4), CD2 molecule (CD2), and amine oxidase copper-containing 2(AOC2)) were identified, and then verified by quantitative real-time PCR (qRT-PCR) in semimembranosus muscles. Additionally, the gene set enrichment analysis and single-sample gene set enrichment analysis of hallmark gene sets were collectively performed on the identified biomarkers. Finally, the transcription factor-biomarker and lncRNA-miRNA-mRNA (biomarker) networks were predicted. The identified potential sex-specific biomarkers may provide new insights into the molecular mechanisms of IMF deposition and the beneficial foundation for improving meat quality in pig breeding.

Expression heterogeneity of ABC-transporter family genes and chemosensitivity genes in gastric tumor, carcinomatosis and lymph node metastases

Introduction. Metastatic tumors (particularly gastric cancer) have been found to be characterized by heterogeneity between the primary tumor and metastases. This type of heterogeneity comes to the fore when treating primary-metastatic forms of tumor and is an important reason for the low effectiveness of their treatment. In this regard, comparative analysis of ABC-transporter gene expression and chemosensitivity genes will allow to characterize to a certain extent the resistance and sensitivity of primary tumor, carcinomatosis and metastases to therapy and provide the basis for personalized treatment approach.Aim. To evaluate expression heterogeneity of ABC-transporter genes and chemosensitivity genes in gastric tumor, carcinomatosis and lymph node metastases.Materials and methods. Overall 41 patients with disseminated gastric cancer stage IV with carcinomatosis of peritoneum were included in the investigation. All patients underwent surgery according to Roux palliative gastrectomy. After surgery patients underwent chemotherapy depending on indications. RNA was isolated using RNeasy Plus mini kit (Qiagen, Germany). The expression level of ABC transporter genes (ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2) and chemosensitivity genes (BRCA1, RRM1, ERCC1, TOP1, TOP2α, TUBβ3, TYMS, GSTP1) was assessed by reverse transcription polymerase chain reaction (RT-PCR) in primary tumor, carcinomatosis and lymph node metastases.Results. The expression levels of the genes under study were shown to vary widely. For ABC transporter genes, ABCG1 (3.1 ± 1.1; max 32.0), ABCG2 (7.9 ± 2.3; max 54.1), ABCG2 (9.6 ± 3.8; max 101.0) were the most expressed genes in gastric tumor tissue, carcinomatosis and lymph node metastasis, respectively. Hyperexpression among chemosensitivity genes at all three sites was characteristic only of TOP2α (17.2 ± 6.0; max. 161.9; 10.8 ± 4.1; max. 105.1; 35.3 ± 0.8; max. 439.6, respectively). We found that TOP2α and BRCA1 gene expression levels were higher in lymph node metastasis compared with gastric tumor tissue and carcinomatosis (at p = 0.005 and p = 0.001). Whereas ABCC1 gene expression was statistically significantly higher in carcinomatosis (p = 0.03).Conclusion. Thus, a high level of expression heterogeneity is observed in gastric cancer, which affects the expression patterns of various genes in different localizations. The expression profile can be used to determine the level of heterogeneity and approach to personalized therapy tactics.

Rearrangement with the nkd2 promoter contributed to allelic diversity of the r1 gene in maize (Zea mays).

SUMMARYThe maize red1 (r1) locus regulates anthocyanin accumulation and is a classic model for allelic diversity; changes in regulatory regions are responsible for most of the variation in gene expression patterns. Here, an intrachromosomal rearrangement between the distal upstream region of r1 and the region of naked endosperm 2 (nkd2) upstream to the third exon generated a nkd2 null allele lacking the first three exons, and the R1‐st (stippled) allele with a novel r1 5′ promoter region homologous to 5′ regions from nkd2‐B73. R1‐sc:124 (an R1‐st derivative) shows increased and earlier expression than a standard R1‐g allele, as well as ectopic expression in the starchy endosperm compartment. Laser capture microdissection and RNA sequencing indicated that ectopic R1‐sc:124 expression impacted expression of genes associated with RNA modification. The expression of R1‐sc:124 resembled nkd2‐W22 expression, suggesting that nkd2 regulatory sequences may influence the expression of R1‐sc:124. The r1‐sc:m3 allele is derived from R1‐sc:124 by an insertion of a Ds6 transposon in intron 4. This insertion blocks anthocyanin regulation by causing mis‐splicing that eliminates exon 5 from the mRNA. This allele serves as an important launch site for Ac/Ds mutagenesis studies, and two Ds6 insertions believed to be associated with nkd2 mutant alleles were actually located in the r1 5′ region. Among annotated genomes of teosinte and maize varieties, the nkd2 and r1 loci showed conserved overall gene structures, similar to the B73 reference genome, suggesting that the nkd2‐r1 rearrangement may be a recent event.

Peering Into Candida albicans Pir Protein Function and Comparative Genomics of the Pir Family.

The fungal cell wall, comprised primarily of protein and polymeric carbohydrate, maintains cell structure, provides protection from the environment, and is an important antifungal drug target. Pir proteins (proteins with internal repeats) are linked to cell wall β-1,3-glucan and are best studied in Saccharomyces cerevisiae. Sequential deletion of S. cerevisiae PIR genes produces strains with increasingly notable cell wall damage. However, a true null mutant lacking all five S. cerevisiae PIR genes was never constructed. Because only two PIR genes (PIR1, PIR32) were annotated in the Candida albicans genome, the initial goal of this work was to construct a true Δpir/Δpir null strain in this species. Unexpectedly, the phenotype of the null strain was almost indistinguishable from its parent, leading to the search for other proteins with Pir function. Bioinformatic approaches revealed nine additional C. albicans proteins that share a conserved Pir functional motif (minimally DGQ). Examination of the protein sequences revealed another conserved motif (QFQFD) toward the C-terminal end of each protein. Sequence similarities and presence of the conserved motif(s) were used to identify a set of 75 proteins across 16 fungal species that are proposed here as Pir proteins. The Pir family is greatly expanded in C. albicans and C. dubliniensis compared to other species and the orthologs are known to have specialized function during chlamydospore formation. Predicted Pir structures showed a conserved core of antiparallel beta-sheets and sometimes-extensive loops that contain amino acids with the potential to form linkages to cell wall components. Pir phylogeny demonstrated emergence of specific ortholog groups among the fungal species. Variation in gene expression patterns was noted among the ortholog groups during growth in rich medium. PIR allelic variation was quite limited despite the presence of a repeated sequence in many loci. Results presented here demonstrate that the Pir family is larger than previously recognized and lead to new hypotheses to test to better understand Pir proteins and their role in the fungal cell wall.

Construction and Validation of Two Hepatocellular Carcinoma-Progression Prognostic Scores Based on Gene Set Variation Analysis.

Background: Liver hepatocellular carcinoma (LIHC) remains a global health challenge with a low early diagnosis rate and high mortality. Therefore, finding new biomarkers for diagnosis and prognosis is still one of the current research priorities. Methods: Based on the variation of gene expression patterns in different stages, the LIHC-development genes (LDGs) were identified by differential expression analysis. Then, prognosis-related LDGs were screened out to construct the LIHC-unfavorable gene set (LUGs) and LIHC-favorable gene set (LFGs). Gene set variation analysis (GSVA) was conducted to build prognostic scoring models based on the LUGs and LFGs. ROC curve analysis and univariate and multivariate Cox regression analysis were carried out to verify the diagnostic and prognostic utility of the two GSVA scores in two independent datasets. Additionally, the key LCGs were identified by the intersection analysis of the PPI network and univariate Cox regression and further evaluated their performance in expression level and prognosis prediction. Single-sample GSEA (ssGSEA) was performed to understand the correlation between the two GSVA enrichment scores and immune activity. Result: With the development of LIHC, 83 LDGs were gradually upregulated and 247 LDGs were gradually downregulated. Combining with LIHC survival analysis, 31 LUGs and 32 LFGs were identified and used to establish the LIHC-unfavorable GSVA score (LUG score) and LIHC-favorable GSVA score (LFG score). ROC curve analysis and univariate/multivariate Cox regression analysis suggested the LUG score and LFG score could be great indicators for the early diagnosis and prognosis prediction. Four genes (ESR1, EHHADH, CYP3A4, and ACADL) were considered as the key LCGs and closely related to good prognosis. The frequency of TP53 mutation and copy number variation (CNV) were high in some LCGs. Low-LFG score patients have active metabolic activity and a more robust immune response. The high-LFG score patients characterized immune activation with the higher infiltration abundance of type I T helper cells, DC, eosinophils, and neutrophils, while the high-LUG score patients characterized immunosuppression with the higher infiltration abundance of type II T helper cells, TRegs, and iDC. The high- and low-LFG score groups differed significantly in immunotherapy response scores, immune checkpoints expression, and IC50 values of common drugs. Conclusion: Overall, the LIHC-progression characteristic genes can be great diagnostic and prognostic signatures and the two GSVA score systems may become promising indices for guiding the tumor treatment of LIHC patients.

Meeting in the Middle: Lessons and Opportunities from Studying C3-C4 Intermediates.

The discovery of C3-C4 intermediate species nearly 50 years ago opened up a new avenue for studying the evolution of photosynthetic pathways. Intermediate species exhibit anatomical, biochemical, and physiological traits that range from C3 to C4. A key feature of C3-C4 intermediates that utilize C2 photosynthesis is the improvement in photosynthetic efficiency compared with C3 species. Although the recruitment of some core enzymes is shared across lineages, there is significant variability in gene expression patterns, consistent with models that suggest numerous evolutionary paths from C3 to C4 photosynthesis. Despite the many evolutionary trajectories, the recruitment of glycine decarboxylase for C2 photosynthesis is likely required. As technologies enable high-throughput genotyping and phenotyping, the discovery of new C3-C4 intermediates species will enrich comparisons between evolutionary lineages. The investigation of C3-C4 intermediate species will enhance our understanding of photosynthetic mechanisms and evolutionary processes and will potentially aid in crop improvement.

An Integrative Single-cell Transcriptomic Atlas of the Post-natal Mouse Mammary Gland Allows Discovery of New Developmental Trajectories in the Luminal Compartment.

The mammary gland is a highly dynamic organ which undergoes periods of expansion, differentiation and cell death in each reproductive cycle. Partly because of the dynamic nature of the gland, mammary epithelial cells (MECs) are extraordinarily heterogeneous. Single cell RNA-seq (scRNA-seq) analyses have contributed to understand the cellular and transcriptional heterogeneity of this complex tissue. Here, we integrate scRNA-seq data from three foundational reports that have explored the mammary gland cell populations throughout development at single-cell level using 10× Chromium Drop-Seq. We center our analysis on post-natal development of the mammary gland, from puberty to post-involution. The new integrated study corresponds to RNA sequences from 53,686 individual cells, which greatly outnumbers the three initial data sets. The large volume of information provides new insights, as a better resolution of the previously detected Procr+ stem-like cell subpopulation or the identification of a novel group of MECs expressing immune-like markers. Moreover, here we present new pseudo-temporal trajectories of MEC populations at two resolution levels, that is either considering all mammary cell subtypes or focusing specifically on the luminal lineages. Interestingly, the luminal-restricted analysis reveals distinct expression patterns of various genes that encode milk proteins, suggesting specific and non-redundant roles for each of them. In summary, our data show that the application of bioinformatic tools to integrate multiple scRNA-seq data-sets helps to describe and interpret the high level of plasticity involved in gene expression regulation throughout mammary gland post-natal development.

Incubation temperature affects yolk utilization through changes in expression of yolk sac tissue functional genes

The yolk sac tissue (YST) is a multifunctional metabolic organ supporting chicken embryonic development. This study examined whether incubation temperatures (ITs) affect YST functions. For this purpose, 300 eggs were assigned to 3 groups and incubated at control IT of 37.8°C, at 1.5°C below, 36.3°C (cold IT), and at 1.5°C above, 39.3°C (hot IT). For each group, 6 embryos' whole body mass and residual yolk (RSY) weights were recorded during incubation, and YST was sampled for both histology and gene expression analysis. YST functionality during incubation was examined by regression analysis, comparing changes in expression patterns of genes involved in lipid uptake and metabolism (LRP2, ApoA1), oligopeptides uptake (PepT1), gluconeogenesis (FBP1), glycogenesis (GYS2), and thyroid hormones regulation (TTR, DIO1, DIO2). Results show that hot and cold ITs affected YST gene expression and yolk utilization. PepT1 expression decreased towards hatch, in both hot and cold ITs, while in the Control IT, it reached a plateau. ApoA1 and DIO2 expression showed a moderate linear fit compared to polynomial fit in the control. GYS2 expression had no change along incubation, while in the control IT, it showed a polynomial fit. Expression of LRP2, FBP1, and DIO1 genes was affected by either cold or hot IT's. TTR expression patterns were similar in all IT groups. The variations in gene expression patterns observed in the 3 ITs can explain the changes in yolk utilization, an important parameter for hatchling quality. While the control IT showed optimal utilization, with an RSY value of 11.12% at the day of hatch, the cold and hot IT groups exhibited lower utilization with an RSY value of 18.18 and 29.99%, respectively. These findings are the first to show that ITs change the expression of key YST genes, leading to variations in yolk utilization by the embryo.

GENE-47. A 3D ATLAS TO EVALUATE THE SPATIAL PATTERNING OF GENETIC ALTERATIONS AND TUMOR CELL STATES IN GLIOMA

Abstract BACKGROUND Previous studies of solid tumors have been restricted in their ability to map how heterogeneous cell populations evolved within the tumor in three-dimensional (3D) space due to insufficient sampling, typically one sample per tumor, and a lack of knowledge of where within the tumor the sample was obtained. Knowledge of the extensivity of heterogeneity and how it is spatially distributed is crucial for assessing whether a therapeutic target is truly tumor-wide, and for exploring how mutations relate to heterogeneity in the local microenvironment. METHODS We developed a novel platform to integrate and visualize in 3D multi-omics data generated from each of 8–10 spatially mapped samples per tumor. Together, the 171 samples collected using this approach from 16 adult diffuse glioma at diagnosis and recurrence form a novel resource – the 3D Glioma Atlas. RESULTS By maximally sampling the tumor geography without excluding samples based on low cancer cell fraction (CCF), we identify a subpopulation of glioblastoma with pervasively lower CCF likely excluded by other atlases, such as the TCGA, that used stringent CCF cutoffs. Exome sequencing of 3D-mapped samples from lower-grade tumors revealed that clonal expansions are typically spatially segregated, implying minimal tumor-wide intermixing of genetically heterogenous cells. Heterogeneity is less spatially segregated for faster-growing high-grade tumors, suggesting that cell populations expand in these tumors differently. Recurrent low-grade tumors have greater intratumoral mutational heterogeneity than newly diagnosed tumors, though this did not translate into greater dissimilarity in gene expression profiles for recurrent tumors, suggesting minimal functional impact of this additional mutational diversity on gene expression. CONCLUSIONS The delineation of spatial patterns of heterogeneity that our work provides enables more informed interpretation of biopsies and greater insight into the factors shaping intratumoral variation of gene expression patterns. Ongoing work is exploring the spatial patterning of amplification events and the tumor microenvironment.