Differential Expression Of Numerous Genes Research Articles

Two sirtuin proteins, Hst3 and Hst4, modulate asexual development, stress tolerance, and virulence by affecting global gene expression in Beauveria bassiana.

Beauveria bassiana is a widely used entomopathogenic fungus in insect biological control applications. In this study, we investigated the role of two sirtuin homologs, BbHst3 and BbHst4, in the biological activities and pathogenicity of B. bassiana. Our results showed that deletion of BbHst3 and/or BbHst4 led to impaired sporulation, reduced (~50%) conidial production, and decreased tolerance to various stresses, including osmotic, oxidative, and cell wall-disturbing agents. Moreover, BbHst4 plays dominant roles in histone H3-K56 acetylation and DNA damage response, while BbHst3 is more responsible for maintaining cell wall integrity. Transcriptomic analyses revealed significant changes (>1,500 differentially expressed genes) in gene expression patterns in the mutant strains, particularly in genes related to secondary metabolism, detoxification, and transporters. Furthermore, the ΔBbHst3, ΔBbHst4, and ΔBbHst3ΔBbHst4 strains exhibited reduced virulence in insect bioassays, with decreased (~20%) abilities to kill insect hosts through topical application and intra-hemocoel injection. These findings highlight the crucial role of BbHst3 and BbHst4 in sporulation, DNA damage repair, cell wall integrity, and fungal infection in B. bassiana. Our study provides new insights into the regulatory mechanisms underlying the biological activities and pathogenicity of B. bassiana and emphasizes the potential of targeting sirtuins for improving the efficacy of fungal biocontrol agents.IMPORTANCESirtuins, as a class of histone deacetylases, have been shown to play important roles in various cellular processes in fungi, including asexual development, stress response, and pathogenicity. By investigating the functions of BbHst3 and BbHst4, we have uncovered their critical contributions to important phenotypes in Beauveria bassiana. Deletion of these sirtuin homologs led to reduced conidial yield, increased sensitivity to osmotic and oxidative stresses, impaired DNA damage repair processes, and decreased fungal virulence. Transcriptomic analyses showed differential expression of numerous genes involved in secondary metabolism, detoxification, transporters, and virulence-related factors, potentially uncovering new targets for manipulation and optimization of fungal biocontrol agents. Our study also emphasizes the significance of sirtuins as key regulators in fungal biology and highlights their potential as promising targets for the development of novel antifungal strategies.

Masson pine pollen aqueous extract ameliorates cadmium-induced kidney damage in rats.

Introduction: Cadmium (Cd) is a hazardous environmental pollutant present in soil, water, and food. Accumulation of Cd in organisms can cause systematic injury and damage to the kidney. The Masson pine pollen aqueous extract (MPPAE) has attracted increasing attention due to its antioxidant activity and ability to enhance immunity. Methods: In this study, we investigated the potential of MPPAE to protect against Cd-induced kidney damage in rats and the underlying mechanism. The transcriptome and metabolome of rats with Cd-induced kidney damage, following treatment with MPPAE, were explored. Results: The concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA) were both significantly altered after treatment with MPPAE. Furthermore, sequencing and analysis of the transcriptome and metabolome of rats with Cd-induced kidney damage, following treatment with MPPAE, revealed differential expression of numerous genes and metabolites compared with the untreated control rats. These differentially expressed genes (DEGs) included detoxification-related genes such as cytochrome P450 and the transporter. The differentially expressed metabolites (DEMs) included 4-hydroxybenzoic acid, L-ascorbate, and ciliatine. Conjoint transcriptome and metabolome analysis showed that several DEGs were correlated with DEMs. Conclusion: These preliminary findings indicate the potential of MPPAE for the treatment of toxic metal poisoning.

Synergistic Effect of Treatment with Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Lipopolysaccharide on the Inflammatory Response of Porcine Pulmonary Microvascular Endothelial Cells.

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) often causes secondary bacterial infection in piglets, resulting in inflammatory lung injury and leading to high mortality rates and significant economic losses in the pig industry. Microvascular endothelial cells (MVECs) play a crucial role in the inflammatory response. Previous studies have shown that HP-PRRSV can infect porcine pulmonary MVECs and damage the endothelial glycocalyx. To further understand the role of pulmonary MVECs in the pathogenesis of HP-PRRSV and its secondary bacterial infection, in this study, cultured porcine pulmonary MVECs were stimulated with a HP-PRRSV HN strain and lipopolysaccharide (LPS). The changes in gene expression profiles were analyzed through transcriptome sequencing, and the differentially expressed genes were verified using qRT-PCR, Western blot, and ELISA. Furthermore, the effects on endothelial barrier function and regulation of neutrophil trans-endothelial migration were detected using the Transwell model. HP-PRRSV primarily induced differential expression of numerous genes associated with immune response, including IFIT2, IFIT3, VCAM1, ITGB4, and CCL5, whereas LPS triggered an inflammatory response involving IL6, IL16, CXCL8, CXCL14, and ITGA7. Compared to the individual effect of LPS, when given after HN-induced stimulation, it caused a greater number of changes in inflammatory molecules, such as VCAM1, IL1A, IL6, IL16, IL17D, CCL5, ITGAV, IGTB8, and TNFAIP3A, a more significant reduction in transendothelial electrical resistance, and higher increase in neutrophil transendothelial migration. In summary, these results suggest a synergistic effect of HP-PRRSV and LPS on the inflammatory response of porcine pulmonary MVECs. This study provides insights into the mechanism of severe lung injury caused by secondary bacterial infection following HP-PRRSV infection from the perspective of MVECs, emphasizing the vital role of pulmonary MVECs in HP-PRRSV infection.

BcTaf14 regulates growth and development, virulence, and stress responses in the phytopathogenic fungus Botrytis cinerea.

TATA box-binding protein (TBP)-associated factor 14 (Taf14), a transcription-associated factor containing a conserved YEATS domain and an extra-terminal (ET) domain, is a multifunctional protein in Saccharomyces cerevisiae. However, the role of Taf14 in filamentous phytopathogenic fungi is not well understood. In this study, the homologue of ScTaf14 in Botrytis cinerea (named BcTaf14), a destructive phytopathogen causing grey mould, was investigated. The BcTaf14 deletion strain (ΔBcTaf14) showed pleiotropic defects, including slow growth, abnormal colony morphology, reduced conidiation, abnormal conidial morphology, reduced virulence, and altered responses to various stresses. The ΔBcTaf14 strain also exhibited differential expression of numerous genes compared to the wild-type strain. BcTaf14 could interact with the crotonylated H3K9 peptide, and mutation of two key sites (G80 and W81) in the YEATS domain disrupted this interaction. The mutation of G80 and W81 affected the regulatory effect of BcTaf14 on mycelial growth and virulence but did not affect the production and morphology of conidia. The absence of the ET domain at the C-terminus rendered BcTaf14 unable to localize to the nucleus, and the defects of ΔBcTaf14 were not recovered to wild-type levels when BcTaf14 without the ET domain was expressed. Our results provide insight into the regulatory roles of BcTaf14 and its two conserved domains in B. cinerea and will be helpful for understanding the function of the Taf14 protein in plant-pathogenic fungi.

Biocontrol of antifungal volatiles produced by Ceriporia lacerate HG2011 against citrus fruit rot incited by Penicillium spp.

Developing effective bio-control agents can safely and effectively reduce the high rate of citrus fruit decay caused by Penicillium spp. in post-harvest storage. Thus, present experiments were conducted to investigate the biocontrol efficiency of volatiles produced by Ceriporia lacerate HG2011 against Penicillium spp., P. italicum infection of wounds artificially created on citrus fruit, and fruit rot in storage. This fungus greatly suppressed the growth and sporulation of P. italicum and P. digitatum in pure culture. Among the 35 volatiles produced by C. lacerate HG2011, 12 were known as antimicrobial substances without toxicity to humans and 6 showed direct inhibitory effects on P. italicum (3 were known as antifungal substances, whilst linoleyl acetate, n-pentadecanol, and 2-nonadecanone have not been reported with antifungal potential in literature). The volatiles induced differential expression of numerous genes in P. italicum, including those responsible for the GO terms involved in the response to toxic substances and pathways SNARE interaction in vesicular transport and synthesis and degradation of ketone bodies. These results implied that the volatiles were toxic to P. italicum and interfered with energy supply, acid-base balance, protein processing, biological macromolecule breakdown, material transport, etc. in pathogen cells. As a result, the control efficiency of the volatiles reached 72 % against P. italicum infection of wounds on citrus fruit and 60 % against fruit rot in storage. Taken together, C. lacerate HG2011 produced multiple antifungal volatiles and interfered with pathogen metabolisms in many ways, showing a promising use in citrus fruit storage.

Adipose Tissue Myeloid-Lineage Neuroimmune Cells Express Genes Important for Neural Plasticity and Regulate Adipose Innervation.

Peripheral nerves allow a bidirectional communication between brain and adipose tissues, and many studies have clearly demonstrated that a loss of the adipose nerve supply results in tissue dysfunction and metabolic dysregulation. Neuroimmune cells closely associate with nerves in many tissues, including subcutaneous white adipose tissue (scWAT). However, in scWAT, their functions beyond degrading norepinephrine in an obese state remain largely unexplored. We previously reported that a myeloid-lineage knockout (KO) of brain-derived neurotrophic factor (BDNF) resulted in decreased innervation of scWAT, accompanied by an inability to brown scWAT after cold stimulation, and increased adiposity after a high-fat diet. These data underscored that adipose tissue neuroimmune cells support the peripheral nerve supply to adipose and impact the tissue’s metabolic functions. We also reported that a subset of myeloid-lineage monocyte/macrophages (Ly6c+CCR2+Cx3cr1+) is recruited to scWAT in response to cold, a process known to increase neurite density in adipose and promote metabolically healthy processes. These cold-induced neuroimmune cells (CINCs) also expressed BDNF. Here we performed RNAseq on CINCs from cold-exposed and room temperature-housed mice, which revealed a striking and coordinated differential expression of numerous genes involved in neuronal function, including neurotrophin signaling and axonal guidance, further supporting that CINCs fulfill a nerve-supporting role in adipose. The increased expression of leukocyte transendothelial migration genes in cold-stimulated CINCs also confirms prior evidence that they are recruited to scWAT and are not tissue resident. We now provide whole-depot imaging of scWAT from LysM-BDNF KO mice, revealing a striking reduction of innervation across the depot fitting with their reduced energy expenditure phenotype. By contrast, Cx3cr1-BDNF KO mice (a macrophage subset of LysM+ cells) exhibited increased thermogenesis and energy expenditure, with compensatory increased food intake and no change in adiposity or body weight. While these KO mice also exhibit a significantly reduced innervation of scWAT, especially around the subiliac lymph node, they displayed an increase in small fiber sympathetic neurite branching, which may underlie their increased thermogenesis. We propose a homeostatic role of scWAT myeloid-lineage neuroimmune cells together in nerve maintenance and neuro-adipose regulation of energy expenditure.

Resistance exercise training increases skeletal muscle mitochondrial respiration in chronic obstructive pulmonary disease

AbstractBackgroundChronic obstructive pulmonary disease (COPD) is associated with skeletal muscle mitochondrial dysfunction. Resistance exercise training (RT) is a training modality with a relatively small pulmonary demand that has been suggested to increase skeletal muscle oxidative enzyme activity in COPD. Whether a shift into a more oxidative profile following RT also translates into increased mitochondrial respiratory capacity in COPD is yet to be established.MethodsThis study investigated the effects of 13 weeks of RT on m. vastus lateralis mitochondrial capacity in 11 persons with moderate COPD [45% females, age: 69 ± 4 years (mean ± SD), predicted forced expiratory volume in 1 s (FEV1): 56 ± 7%] and 12 healthy controls (75% females, age: 66 ± 5 years, predicted FEV1: 110 ± 16%). RT was supervised and carried out two times per week. Leg exercises included leg press, knee extension, and knee flexion and were performed unilaterally with one leg conducting high‐load training (10 repetitions maximum, 10RM) and the other leg conducting low‐load training (30 repetitions maximum, 30RM). One‐legged muscle mass, maximal muscle strength, and endurance performance were determined prior to and after the RT period, together with mitochondrial respiratory capacity using high‐resolution respirometry and citrate synthase (CS) activity (a marker for mitochondrial volume density). Transcriptome analysis of genes associated with mitochondrial function was performed.ResultsResistance exercise training led to similar improvements in one‐legged muscle mass, muscle strength, and endurance performance in COPD and healthy individuals. In COPD, mitochondrial fatty acid oxidation capacity and oxidative phosphorylation increased following RT (+13 ± 22%, P = 0.033 and +9 ± 23%, P = 0.035, respectively). Marked increases were also seen in COPD for mitochondrial volume density (CS activity, +39 ± 35%, P = 0.001), which increased more than mitochondrial respiration, leading to lowered intrinsic mitochondrial function (respiration/CS activity) for complex‐1‐supported respiration (−12 ± 43%, P = 0.033), oxidative phosphorylation (−10 ± 42%, P = 0.037), and electron transfer system capacity (−6 ± 52%, P = 0.027). No differences were observed between 10RM and 30RM RT, nor were there any adaptations in mitochondrial function following RT in healthy controls. RT led to differential expression of numerous genes related to mitochondrial function in both COPD and healthy controls, with no difference being observed between groups.ConclusionsThirteen weeks of RT resulted in augmented skeletal muscle mitochondrial respiratory capacity in COPD, accompanied by alterations in the transcriptome and driven by an increase in mitochondrial quantity rather than improved mitochondrial quality.

Behavioral and Gene Regulatory Responses to Developmental Drug Exposures in Zebrafish.

Developmental consequences of prenatal drug exposure have been reported in many human cohorts and animal studies. The long-lasting impact on the offspring—including motor and cognitive impairments, cranial and cardiac anomalies and increased prevalence of ADHD—is a socioeconomic burden worldwide. Identifying the molecular changes leading to developmental consequences could help ameliorate the deficits and limit the impact. In this study, we have used zebrafish, a well-established behavioral and genetic model with conserved drug response and reward pathways, to identify changes in behavior and cellular pathways in response to developmental exposure to amphetamine, nicotine or oxycodone. In the presence of the drug, exposed animals showed altered behavior, consistent with effects seen in mammalian systems, including impaired locomotion and altered habituation to acoustic startle. Differences in responses seen following acute and chronic exposure suggest adaptation to the presence of the drug. Transcriptomic analysis of exposed larvae revealed differential expression of numerous genes and alterations in many pathways, including those related to cell death, immunity and circadian rhythm regulation. Differential expression of circadian rhythm genes did not correlate with behavioral changes in the larvae, however, two of the circadian genes, arntl2 and per2, were also differentially expressed at later stages of development, suggesting a long-lasting impact of developmental exposures on circadian gene expression. The immediate-early genes, egr1, egr4, fosab, and junbb, which are associated with synaptic plasticity, were downregulated by all three drugs and in situ hybridization showed that the expression for all four genes was reduced across all neuroanatomical regions, including brain regions implicated in reward processing, addiction and other psychiatric conditions. We anticipate that these early changes in gene expression in response to drug exposure are likely to contribute to the consequences of prenatal exposure and their discovery might pave the way to therapeutic intervention to ameliorate the long-lasting deficits.

Novel Antifungal Activity of Q-Griffithsin, a Broad-Spectrum Antiviral Lectin.

ABSTRACTThere is a rising global incidence of Candida strains with high levels of resistance to fluconazole and other antifungal drugs, hence the need for novel antifungal treatment strategies. Here, we describe the first evidence of antifungal activity of Q-Griffithsin (Q-GRFT), a recombinant oxidation-resistant variant of Griffithsin, a marine red algal lectin with broad-spectrum antiviral activity. We demonstrated that Q-GRFT binds to α-mannan in the Candida albicans cell wall. We also observed that Q-GRFT binding disrupted cell wall integrity and induced reactive oxidative species (ROS) formation, resulting in cell death. Furthermore, we showed that Q-GRFT inhibited the growth of other Candida species C. glabrata, C. parapsilosis, and C. krusei and had modest activity against some strains of multi- and pandrug-resistant C. auris. We found that Q-GRFT induced differential expression of numerous genes involved in response to cell stress, including those responsible for neutralizing ROS production and cell cycle regulation. In conclusion, this novel antifungal activity suggests that Q-GRFT is potentially an ideal drug candidate and represents an alternative strategy for the prevention and treatment of candidiasis.IMPORTANCE Fungal infections contribute to morbidity and mortality annually, and the number of organisms that are nonresponsive to the current available drug regimens are on the rise. There is a need to develop new agents to counter these infections and to add to the limited arsenal available to treat fungal infections. Our study has identified Q-GRFT, a broad-spectrum antiviral protein that harbors growth-inhibitory activity against several Candida strains, as a potential candidate for the prevention and treatment of fungal infections.

Two citrus KNAT-like genes, CsKN1 and CsKN2, are involved in the regulation of spring shoot development in sweet orange.

Shoot-tip abortion is a very common phenomenon in some perennial woody plants and it affects the height, architecture, and branch orientation of trees; however, little is currently known about the underlying mechanisms. In this study, we identified a gene in sweet orange (Citrus sinensis) encoding a KNAT-like protein (CsKN1) and found high expression in the shoot apical meristem (SAM). Overexpression of CsKN1 in transgenic plants prolonged the vegetative growth of SAMs, whilst silencing resulted in either the loss or inhibition of SAMs. Yeast two-hybrid analysis revealed that CsKN1 interacted with another citrus KNAT-like protein (CsKN2), and overexpression of CsKN2 in lemon and tobacco caused an extreme multiple-meristem phenotype. Overexpression of CsKN1 and CsKN2 in transgenic plants resulted in the differential expression of numerous genes related to hormone biosynthesis and signaling. Yeast one-hybrid analysis revealed that the CsKN1-CsKN2 complex can bind to the promoter of citrus floral meristem gene LEAFY (CsLFY) and inhibit its expression. These results indicated that CsKN1 might prolong the vegetative growth period of SAMs by delaying flowering. In addition, an ethylene-responsive factor (CsERF) was found to bind to the CsKN1 promoter and suppresses its transcription. Overexpression of CsERF in Arabidopsis increased the contents of ethylene and reactive oxygen species, which might induce the occurrence of shoot-tip abscission. On the basis of our results, we conclude that CsKN1 and CsKN2 might work cooperatively to regulate the shoot-tip abscission process in spring shoots of sweet orange.

A drought-responsive rice amidohydrolase is the elusive plant guanine deaminase with the potential to modulate the epigenome.

Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S‐adenosyl‐methionine (SAM) to xanthine pathway. SAM is converted to S‐adenosyl‐homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.

Transcriptome based functional identification and application of regulator AbrB on alkaline protease synthesis in Bacillus licheniformis 2709

Bacillus licheniformis 2709 is the major alkaline protease producer, which has great potential value of industrial application, but how the high-producer can be regulated rationally is still not completely understood. It's meaningful to understand the metabolic processes during alkaline protease production in industrial fermentation medium. Here, we collected the transcription database at various enzyme-producing stages (preliminary stage, stable phase and decline phase) to specifically research the synthesized and regulatory mechanism of alkaline protease in B. licheniformis. The RNA-sequencing analysis showed differential expression of numerous genes related to several processes, among which genes correlated with regulators were concerned, especially the major differential gene abrB on enzyme (AprE) synthesis was investigated. It was further verified that AbrB is a repressor of AprE by plasmid-mediated over-expression due to the severely descending enzyme activity (11,300 U/mL to 2695 U/mL), but interestingly it is indispensable for alkaline protease production because the enzyme activity of the null abrB mutant was just about 2279 U/mL. Thus, we investigated the aprE transcription by eliminating the theoretical binding site (TGGAA) of AbrB protein predicated by computational strategy, which significantly improved the enzyme activity by 1.21-fold and gene transcription level by 1.77-fold in the mid-log phase at a cultivation time of 18 h. Taken together, it is of great significance to improve the production strategy, control the metabolic process and oriented engineering by rational molecular modification of regulatory network based on the high throughput sequencing and computational prediction.

FcRγ Gene Editing Reprograms Conventional NK Cells to Display Key Features of Adaptive Human NK Cells.

SummaryAdaptive human natural killer (NK) cells display significantly enhanced responsiveness to a broad-range of antibody-bound targets through the engagement of CD16 compared to conventional NK cells, yet direct reactivity against tumor targets is generally reduced. Adaptive NK cells also display a distinct phenotype and differential expression of numerous genes, including reduced expression of signaling adapter FcRγ and transcription factor PLZF. However, it is unclear whether differential expression of specific genes is responsible for the characteristics of adaptive NK cells. Using CRISPR-Cas9, we show deletion of FcRγ in conventional NK cells led to enhanced CD16 responsiveness, abolished cell surface expression of natural cytotoxicity receptors, NKp46 and NKp30, and dramatically reduced responsiveness to K562 and Raji tumor cells. However, deletion of PLZF had no notable effects. These results suggest multiple roles for FcRγ and identify its deficiency as an important factor responsible for the functional and phenotypic characteristics exhibited by adaptive NK cells.

Alpha sinuclein expression in blood and its relationship with chronic constipation in a population from Bogotá, D.C., with problems of alcohol consumption

ResumenIntroducción. El consumo excesivo de alcohol resulta en neuroadaptación, neurodegeneración y expresión diferencial de numerosos genes.Objetivo. Determinar la relación entre la expresión del gen de la alfa sinucleína (SNCA) en sangre, las variantes de nucleótido único (Single Nucleotide Variant, SNV) en su región promotora y el estreñimiento crónico en personas con problemas de consumo de alcohol.Materiales y métodos. La muestra estuvo conformada por 35 controles y 27 casos, seleccionados según el puntaje obtenido con la herramienta AUDIT. En el diagnóstico del estreñimiento se aplicaron los criterios de Roma IV. La extracción de ácidos nucleicos se hizo a partir de sangre periférica y se evaluó la expresión del gen mediante qPCR, la cuantificación proteica por ELISA y la presencia de SNV en la región promotora del gen por la secuenciación de Sanger.Resultados. Se observó sobreexpresión génica relativa de ARNm del gen SNCA en el grupo de casos sin relación con el estreñimiento crónico. Se evidenció un riesgo 4,8 veces mayor de presentar estreñimiento en el grupo de casos. Se encontraron nueve variantes de nucleótido simple en un segmento de la región promotora del gen rica en secuencias reguladoras CpG, con frecuencia similar entre los grupos, y se detectó una variante en la posición -2171 que no se encuentra reportada en GenBank para variantes clínicas y cuyo genotipo A/T se relacionó con el incremento de la expresión del ARNm del SNCA.Conclusión. En personas con problemas de consumo de alcohol se evidenció la sobreexpresión del ARNm de alfa sinucleína, lo cual no se relacionó con el diagnóstico de estreñimiento crónico.

Molecular networks of hypoxia and neuronal apoptosis in the cochlea

In organotypic cultures, the modiolus (MOD) region of newborn rats shows afourfold higher rate of cell death than the organ of Corti (OC). The differing vulnerability of OC and MOD cells is related to differential expression of numerous genes (DEG). Organotypic cultures of OC and MOD of 3-5-day-old rats were exposed to anormoxic or a hypoxic (pO2 10-20 mmHg; 5 h) atmosphere. Cell death rate and gene expression as detected by c‑DNA microarray analysis were determined 24 h after the culture was created. Genes with modified expression (n = 60) were analyzed for biological processes according to the DAVID Gene Ontology Database (GO). Molecular networks were created using the STRING and ConsensusPathDB databases. The network of the GO annotations "hypoxia", "inflammation", and "mechanical stimulus" indicates the existence of two gene clusters: acluster with pro-inflammatory genes (Ccl3, Cxcl2, Cxcr4, Ccl20) and acluster with hypoxia-associated genes (e.g., c-Jun, Hif1a, and Vegfa). The network of the GO annotations "positive and negative regulation of neuron apoptotic process" suggests that the differential expression of c-Jun, Ngfr, and Casp3 is important for regulation of programmed cell death in neuronal cells of the OC and MOD. While c‑JUN acts as an important modulator of the balance between cell death and survival, the associations of NGFR and CASP3 seem to be significant for the initiation of cell death. The evaluation and application of findings from biostatistical databases is important for understanding the function of individual genes and gene clusters in medically relevant biological processes.

Alteration of TAC1 expression in Prunus species leads to pleiotropic shoot phenotypes

Prunus persica (peach) trees carrying the “Pillar” or “Broomy” trait (br) have vertically oriented branches caused by loss-of-function mutations in a gene called TILLER ANGLE CONTROL 1 (TAC1). TAC1 encodes a protein in the IGT gene family that includes LAZY1 and DEEPER ROOTING 1 (DRO1), which regulate lateral branch and root orientations, respectively. Here we found that some of the native TAC1 alleles in the hexaploid plum species Prunus domestica, which has a naturally more upright stature, contained a variable length trinucleotide repeat within the same exon 3 region previously found to be disrupted in pillar peach trees. RNAi silencing of TAC1 in plum resulted in trees with severely vertical branch orientations similar to those in pillar peaches but with an even narrower profile. In contrast, PpeTAC1 overexpression in plum led to trees with wider branch angles and more horizontal branch orientations. Pillar peach trees and transgenic plum lines exhibited pleiotropic phenotypes, including differences in trunk and branch diameter, stem growth, and twisting branch phenotypes. Expression profiling of pillar peach trees revealed differential expression of numerous genes associated with biotic and abiotic stress, hormone responses, plastids, reactive oxygen, secondary, and cell wall metabolism. Collectively, the data provide important clues for understanding TAC1 function and show that alteration of TAC1 expression may have broad applicability to agricultural and ornamental tree industries.

Abstract 3957: Characterization of functional heterogeneity and in vivo dynamics of clonal cell populations derived from the triple-negative breast cancer cell line MDA-MB-468

Abstract Tumors evolve through progressive accumulation of (epi)genetic alterations, favoring expansion of the fittest cell populations as a result of stimuli and selective pressures in the microenvironment. Intratumor heterogeneity and the interplay of tumor cells with the microenvironment present an order of complexity that can have profound effects on tumor progression and drug sensitivity. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of functional heterogeneity within a single tumor, and the dynamics and spatial organization of distinct cell populations over time. The overall objective of this study is to characterize the functional heterogeneity of breast tumor cells and evaluate the dynamics of clonal populations within mixtures during tumor progression. We have generated and characterized 31 clonal cell populations (SCP) from the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic heterogeneity with respect to morphology, proliferation rate, survival in suspension, colony formation in soft agar, and tumorigenicity. We have also performed whole-exome sequencing for 7 SCPs, and RNA-seq and RPPA analysis for all SCPs and the parental cell line. These analyses revealed SCP-unique single nucleotide variants, and differential expression of numerous genes. To study the dynamics of SCP mixtures during the development of xenograft tumors, we have transduced 22 SCPs with unique DNA barcodes derived from the ClonTracer DNA barcode library. The 22 barcoded SCPs were mixed in equal proportions and subsequently injected into the mammary fat pad of NOD/SCID mice. Tumors and lungs were collected at different time points (3 weeks, 2 months and 4 months) to determine the relative changes in clonal representation during tumor progression by next generation sequencing. We identified highly reproducible patterns of clonal expansion, with SCP01 and SCP03 being temporarily enriched for in tumor samples collected at 3 weeks and 2 months respectively, and SCP32 becoming progressively enriched for in tumors over time. In addition, we found recurrent enrichment for SCP01 in most lung samples. Moreover, in contrast to the tumor samples, we found a slight enrichment for SCP13, but not SCP32, during in vitro propagation of the SCP mixture. Together these results suggest the existence of distinct competitive advantages of individual clonal populations within certain spatial and temporal windows. In addition, the discordance in SCP dynamics between the in vivo and in vitro arms suggests that enrichment for SCP01, SCP03 and SCP32 is the result of stimuli or selective pressures that are specific to the tumor microenvironment. Guided by the RNA-seq and RPPA analyses, we are currently testing several hypotheses that may explain the observed enrichment patterns. Citation Format: Hendrik J. Kuiken, Sabin Dhakal, Laura M. Selfors, Jett P. Crowdis, Hyo-eun C. Bhang, Frank Stegmeier, Gordon B. Mills, Joan S. Brugge. Characterization of functional heterogeneity and in vivo dynamics of clonal cell populations derived from the triple-negative breast cancer cell line MDA-MB-468 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2017-3957

Survival analysis and microarray profiling identify Cd40 as a candidate for the Salmonella susceptibility locus, Ity5.

The outcome of infection with Salmonella Typhimurium in mouse models of human typhoid fever is dependent upon a coordinated complex immune response. A panel of recombinant congenic strains (RCS) derived from reciprocal backcross of A/J and C57BL/6J mice was screened for their susceptibility to Salmonella infection and two susceptibility loci, Ity4 (Immunity to Typhimurium locus 4) and Ity5, were identified. We validated Ity5 in a genetic environment free of the impact of Ity4 using a cross between A/J and 129S6. Using a time-series analysis of genome-wide transcription during infection, comparing A/J with AcB60 mice having a C57BL/6J-derived Ity5 interval, we have identified the differential expression of the positional candidate gene Cd40, Cd40-associated signaling pathways, and the differential expression of numerous genes expressed in neutrophils. CD40 is known to coordinate T cell-dependent B-cell responses and myeloid cell activation. In fact, CD40 signaling is altered in A/J mice as seen by impaired IgM upregulation during infection, decreased Ig class switching, neutropenia, reduced granulocyte recruitment in response to infection and inflammation, and decreased ERK1/2 activity. These results suggest that altered CD40 signaling and granulocyte recruitment in response to infection are responsible for the Ity5-associated Salmonella susceptibility of A/J mice.

Serglycin protects against high fat diet-induced increase in serum LDL in mice.

Proteoglycans have been implicated in regulation of lipoprotein metabolism. However, the impact of serglycin, the major proteoglycan expressed by many hematopoietic- and endothelial cells, on lipoprotein metabolism has not been explored. Here we addressed this issue by comparing several parameters of lipid metabolism in wild type (WT) and serglycin-/- mice, both at baseline and after feeding mice the Paigen diet. We show that, after feeding this diet for 20 weeks, serglycin deficient mice exhibited elevated concentrations of serum LDL in comparison with WT mice, thus suggesting that serglycin protects against an elevation of serum LDL levels after intake of a high-fat diet. Body weight increased in both groups, but only significantly in the serglycin-/- group. To explore the mechanism underlying this phenotype, genome-wide expression analysis was performed on liver tissues from WT and serglycin-/- mice. This analysis showed that serglycin-deficiency is associated with differential expression of numerous genes involved in the regulation of lipid metabolism, suggesting that the impact of serglycin on LDL levels may be related to effects at the gene expression level. In particular, several members of the CYP gene family were differently regulated in serglycin-/- compared with WT mice. Moreover, upstream regulator analysis suggested that several pro-inflammatory pathways, including the NFκB pathway, could contribute to the impact of serglycin on LDL. Hence, the elevation of serum LDL seen in serglycin-/- mice may be linked to dysregulated inflammatory responses. Taken together, our findings introduce serglycin as a novel player in processes that regulate lipid metabolism.

We can't all be supermodels: the value of comparative transcriptomics to the study of non-model insects.

Insects are the most diverse group of organisms on the planet. Variation in gene expression lies at the heart of this biodiversity and recent advances in sequencing technology have spawned a revolution in researchers' ability to survey tissue-specific transcriptional complexity across a wide range of insect taxa. Increasingly, studies are using a comparative approach (across species, sexes and life stages) that examines the transcriptional basis of phenotypic diversity within an evolutionary context. In the present review, we summarize much of this research, focusing in particular on three critical aspects of insect biology: morphological development and plasticity; physiological response to the environment; and sexual dimorphism. A common feature that is emerging from these investigations concerns the dynamic nature of transcriptome evolution as indicated by rapid changes in the overall pattern of gene expression, the differential expression of numerous genes with unknown function, and the incorporation of novel, lineage-specific genes into the transcriptional profile.