Drive Gene Expression Research Articles

Hepatic transcriptome signature correlated with HOMA-IR explains early nonalcoholic fatty liver disease pathogenesis

Introduction and objectivesNon-alcoholic fatty liver disease (NAFLD) is multistage with heterogeneous outcomes. We studied the influence of insulin resistance (IR) on the hepatic transcriptome of early NAFLD stages, to understand disease development. Materials and methodsIn this cross-sectional study, possible clinicopathological risk factors were compared between mild-NAFL (N = 72) and non-alcoholic steatohepatitis (NASH; N = 51) patients. Liver tissue-transcriptome difference was studied between a subset of 25 mild-NAFL and 20 NASH biopsies and validated on another subset of 12 mild-NAFL and 13 NASH biopsies, using RT-PCR. The relationship between IR driven gene expression changes with fibrosis in NASH was investigated. ResultsSignificantly higher weight (p = 0.005) and elevated levels of HbA1c (p = 0.009), FBG (p = 0.03) and HOMA-IR (p = 0.009) were found in NASH patients. Five differentially expressed genes (DEGs, fold change > 1.5) were identified in NASH-FABP4, FABP5L2, CD24, PRAP1, and SPP1. The DEGs were positively associated with disease severity and HOMA-IR, and were found to be efficient classifiers of mild-NAFL and NASH. Additional 1218 genes identified related to IR (IrCGs), which can classify NASH-with-fibrosis patients separately from mild-NAFL and NASH patients. IrCGs can promote intra-hepatic fat accumulation, dysregulation of the lipid metabolism, lipotoxicity, and activation of cell survival pathways including activation of cell proliferation and differentiation pathways. ConclusionsHepatic expression of genes associated with insulin resistance may drive NAFLD development and progression.

Mouse intestinal tuft cells express advillin but not villin

Tuft (or brush) cells are solitary chemosensory cells scattered throughout the epithelia of the respiratory and alimentary tract. The actin-binding protein villin (Vil1) is used as a marker of tuft cells and the villin promoter is frequently used to drive expression of the Cre recombinase in tuft cells. While there is widespread agreement about the expression of villin in tuft cells there are several disagreements related to tuft cell lineage commitment and function. We now show that many of these inconsistencies could be resolved by our surprising finding that intestinal tuft cells, in fact, do not express villin protein. Furthermore, we show that a related actin-binding protein, advillin which shares 75% homology with villin, has a tuft cell restricted expression in the gastrointestinal epithelium. Our study identifies advillin as a marker of tuft cells and provides a mechanism for driving gene expression in tuft cells but not in other epithelial cells of the gastrointestinal tract. Our findings fundamentally change the way we identify and study intestinal tuft cells.

A social encounter drives gene expression changes linked to neuronal function, brain development, and related disorders in mice expressing the serotonin transporter Ala56 variant

Multiple lines of evidence implicate the serotonin (5-HT) system in social function, including biomarker findings in autism spectrum disorder. In mice, knock-in of a rare Gly56Ala substitution in the serotonin transporter (SERT) causes elevated whole blood 5-HT levels, increased 5-HT clearance in the brain, and altered social and repetitive behavior. To further examine the molecular impact of this variant on social response, SERT Ala56 mutant mice and wildtype littermate controls were exposed to a social or non-social stimulus. We examined the differential activation of the prefrontal cortex, lateral amygdala, and medial amygdala, to social stimuli through RNA sequencing. Differentially expressed genes were enriched in axonal guidance signaling pathways, networks related to nervous system development and function, neurological and psychiatric disorders, and behavior. These identified pathways and networks may shed light on the molecular cascades underlying the impact of altered SERT function on social behavior.

Mapping the Minimal Promoter Sequence for Human Prolidase (Peptidase D; PEPD‐ gene)

Collagen, the most abundant protein in humans, is the primary component of the extracellular matrix. Collagen is also a critical reservoir of proline that, along with hydroxyproline, comprise one‐fourth of the amino acid composition of collagen. During collagen turnover, sequential action of matrix metalloproteinases and peptidases convert collagen into dipeptides. In the subsequent final rate‐limiting step, prolidase (PEPD) hydrolyzes dipeptides containing C‐terminal proline or hydroxyproline into the constituent amino acids, which are then recycled towards collagen synthesis. Due to its critical role in collagen turnover and protein metabolism, prolidase is a key player in several physiological and pathological processes, including wound healing, cell proliferation, inflammation, and carcinogenesis. Consequently, alterations in prolidase expression can profoundly affect normal physiological processes and cause pathological changes. The goal of this study is to narrow down such knowledge gaps, especially, in our understanding of the regulation of PEPD transcription. First, towards determining a functionally active promoter region of PEPD (PEPDp), we cloned a 1587 base pair (bp) DNA region—spanning 1537 bp upstream, and 50 bp downstream, of the transcription start site (TSS)—of PEPD gene into a firefly luciferase reporter plasmid. We confirmed that the PEPDp is functionally active by demonstrating its ability to robustly drive the luciferase expression in the HEK293T cell line. Second, we sought to determine the minimal region of PEPDp that is sufficient and necessary to drive gene expression. We subcloned two progressively shorter deletion fragments of PEPDp—537 bp and 387 bp long into the firefly luciferase reporter system and then compared their promoter activity with that of the full‐length PEPDp. Our findings thus indicate that the lack of any cancellation in the promoter activity of the PEPDp deletion fragments points out that the functionally active region of PEPDp spans ~350 bp upstream and 50 bp downstream of the transcriptional start site (TSS), thereby advancing our knowledge of the molecular mechanisms regulating prolidase expression.Support or Funding InformationThis work is partly supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

Scaling up single-cell mechanics to multicellular tissues - the role of the intermediate filament-desmosome network.

Cells and tissues sense, respond to and translate mechanical forces into biochemical signals through mechanotransduction, which governs individual cell responses that drive gene expression, metabolic pathways and cell motility, and determines how cells work together in tissues. Mechanotransduction often depends on cytoskeletal networks and their attachment sites that physically couple cells to each other and to the extracellular matrix. One way that cells associate with each other is through Ca2+-dependent adhesion molecules called cadherins, which mediate cell-cell interactions through adherens junctions, thereby anchoring and organizing the cortical actin cytoskeleton. This actin-based network confers dynamic properties to cell sheets and developing organisms. However, these contractile networks do not work alone but in concert with other cytoarchitectural elements, including a diverse network of intermediate filaments. This Review takes a close look at the intermediate filament network and its associated intercellular junctions, desmosomes. We provide evidence that this system not only ensures tissue integrity, but also cooperates with other networks to create more complex tissues with emerging properties in sensing and responding to increasingly stressful environments. We will also draw attention to how defects in intermediate filament and desmosome networks result in both chronic and acquired diseases.

Splicing, Mutation, and Methylation Alterations Drive Gene Expression in HPV-OPC more than Copy Number Variation: A Network Propagation Analysis

Splicing, Mutation, and Methylation Alterations Drive Gene Expression in HPV-OPC more than Copy Number Variation: A Network Propagation Analysis

Noncanonical Wnt planar cell polarity signaling in lung development and disease.

The planar cell polarity (PCP) signaling pathway is a potent developmental regulator of directional cell behaviors such as migration, asymmetric division and morphological polarization that are critical for shaping the body axis and the complex three-dimensional architecture of tissues and organs. PCP is considered a noncanonical Wnt pathway due to the involvement of Wnt ligands and Frizzled family receptors in the absence of the beta-catenin driven gene expression observed in the canonical Wnt cascade. At the heart of the PCP mechanism are protein complexes capable of generating molecular asymmetries within cells along a tissue-wide axis that are translated into polarized actin and microtubule cytoskeletal dynamics. PCP has emerged as an important regulator of developmental, homeostatic and disease processes in the respiratory system. It acts along other signaling pathways to create the elaborately branched structure of the lung by controlling the directional protrusive movements of cells during branching morphogenesis. PCP operates in the airway epithelium to establish and maintain the orientation of respiratory cilia along the airway axis for anatomically directed mucociliary clearance. It also regulates the establishment of the pulmonary vasculature. In adult tissues, PCP dysfunction has been linked to a variety of chronic lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary arterial hypertension, stemming chiefly from the breakdown of proper tissue structure and function and aberrant cell migration during regenerative wound healing. A better understanding of these (impaired) PCP mechanisms is needed to fully harness the therapeutic opportunities of targeting PCP in chronic lung diseases.

The endogenous EF-1α promoter is highly active in driving gene overexpression in developing embryos of the Pacific oyster Crassostrea gigas

Gain and loss-of-function analyses are powerful genetic approaches to uncover gene functions in basic and applied research. However, the genetic analysis in bivalve molluscs has been challenged by the lack of effective gene promoters for driving gene overexpression. The cosmopolitan Pacific oyster (Crassostrea gigas) is an economically important marine bivalve and a representative model species for ecological, evolutionary, and developmental studies. Here, we isolated the elongation factor1-α (EF-1α) gene promoter from the Pacific oyster and compared the promoter activity with the commonly used cytomegalovirus (CMV) and Xenopus EF-1α promoters in oyster embryos. We found that the 3465 bp DNA sequence prior to the ATG start codon in oyster EF-1α was able to drive enhanced green fluorescent protein (EGFP) expression in oyster embryos. In contrast, CMV and Xenopus EF-1α promoters failed to direct EGFP expression in oyster embryos. It appeared that the first intron sequence in oyster EF-1α gene was required for the promoter activity. DNA construct without the first intron located upstream of the ATG start codon failed to drive EGFP expression in oyster embryos. Collectively, these studies indicate that the endogenous EF-1α promoter is preferred promoter for driving gene expression in bivalves.

Advancements in AAV-mediated Gene Therapy for Pompe Disease.

Pompe disease (glycogen storage disease type II) is caused by mutations in acid α-glucosidase (GAA) resulting in lysosomal pathology and impairment of the muscular and cardio-pulmonary systems. Enzyme replacement therapy (ERT), the only approved therapy for Pompe disease, improves muscle function by reducing glycogen accumulation but this approach entails several limitations including a short drug half-life and an antibody response that results in reduced efficacy. To address these limitations, new treatments such as gene therapy are under development to increase the intrinsic ability of the affected cells to produce GAA. Key components to gene therapy strategies include the choice of vector, promoter, and the route of administration. The efficacy of gene therapy depends on the ability of the vector to drive gene expression in the target tissue and also on the recipient’s immune tolerance to the transgene protein. In this review, we discuss the preclinical and clinical studies that are paving the way for the development of a gene therapy strategy for patients with early and late onset Pompe disease as well as some of the challenges for advancing gene therapy.

Structurally-Discovered KLF4 Variants Accelerate and Stabilize Reprogramming to Pluripotency

Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domain enable next-generation reprogramming technology with engineered reprogramming factors.

Transcriptional Profiling Reveals Strong Impact of Major Molecular Disease Subgroups and Mixed Epistasis in Chronic Lymphocytic Leukemia

While recurrent mutations in CLL have been extensively catalogued, how driver mutations affect disease phenotypes remains incompletely understood. To address this, we performed RNA sequencing on 184 CLL patient samples and linked gene expression changes to molecular subgroups, gene mutations and copy number variants. Library preparation was performed according to the Illumina TruSeq RNA sample preparation v2 protocol. Samples were paired-end sequenced and two to three samples were multiplexed per lane on Illumina HiSeq 2000, Illumina HiSeq3000/4000 or Illumina HiSeqX machines. Raw RNA-seq reads were demultiplexed and quality control was performed using FastQC version 0.11.5. Internal trimming with STAR version 2.5.2a was used to remove adapters before mapping. Mapping was performed using STAR version 2.5.2a against the Ensembl human reference genome release 75 (Homo sapiens GRCh37.75). STAR was run in default mode with internal adapter trimming using the clip3pAdapterSeq option. Mapped reads were summarized into counts using htseq-count version 0.9.0 with default parameters and union mode. Thus, only fragments unambiguously overlapping with one gene were counted. The count data were then imported into R (version 3.4) for subsequent analysis. We identified robust and previously unknown gene expression signatures associated with recurrent copy number variants (including trisomy 12, del11q22.3, del17p13, del18p12 and gain8q24), gene mutations (TP53, BRAF and SF3B1) and the mutation status of the immunoglobulin heavy-chain variable region (IGHV). The most profound gene expression changes were associated with IGHV, methylation groups and trisomy 12. We found evidence for a significant influence of CNVs beyond the gene dosage effect. In line with these observations, unsupervised clustering showed that these major biological subgroups form distinct clusters and are discernible by unsupervised clustering (IGHV, methylation groups and trisomy 12). We found 3275 genes significantly differentially expressed between M-CLL and U-CLL after adjustment for multiple testing using the method of Benjamini and Hochberg for FDR = 1% . In total 9.5 % of variance within gene expression was associated with the IGHV status. These data suggest a much larger impact on transcriptional changes than previously detected (Ferreira et al. 2014), a finding much more in line with the key impact of IGHV on clinical course and biology of disease. We found distinct expression pattern of up- and downregulated genes for trisomy 12 samples. Even though many upregulated genes are located on chromosome 12, the majority of differentially expressed genes are indeed distributed among the other chromosomes and cannot be therefore not be ascribed to a simple gene dosage effect. To investigate the role of genetic interactions, we tested the collaborative effect on gene expression phenotypes. We investigated epistatic gene expression changes for IGHV status and trisomy 12. Epistasis was defined as a non-linear effect on gene expression between sample with both variants co-occuring and the single variants alone. In total 893 genes showed specific expression pattern in a combined genotype (padj<0.1). These expression changes differed from the expected change by simple combination of the single variant's effects. We observed different ways of epistatic interaction and clustered genes by them. In total, we identified five cluster of genes representing different ways of mixed epistasis as inversion down, suppression, different degrees of buffering and inversion up. To further investigate this interaction we used enrichment tests for genes in the different mixed epistasis cluster. We found genes upregulated in trisomy12 U-CLL sample, but suppressed in M-CLL trisomy12 samples were enriched in Wnt beta catenin and Notch signaling. In summary, our study provides a comprehensive reference data set for gene expression in CLL. We show that IGHV mutation status, recurrent gene mutations and CNVs drive gene expression in a previously underappreciated fashion. This includes epistatic interaction between trisomy 12 and IGHV. Using a novel way to describe coordinated changes we can group genes into sets related to buffering, inversion and suppression. Disclosures Sellner: Takeda: Employment.

Surface Density of CAR Molecules May Modulate the Kinetics of CAR-T Cells In Vivo

Introduction: The various chimeric antigen receptor (CAR) genes used in most CAR-T clinical trials are expressed via the EF1α promoter. Recent reports have suggested that CAR expression using the myeloproliferative sarcoma virus enhancer, with the negative control region deleted and the dl587 Rev-binding site substituted (MND) promoter, to drive CAR expression results in a higher efficiency of gene transduction. The use of a different promoter for CAR expression is expected to alter the expression levels and the surface densities of CAR in transduced T cells. It has been reported previously that while only a few ligands are required to activate TCR signaling, CAR-T cells require approximately 100 CD19 molecules to trigger cytotoxic killing. Here we generated two CAR-T vectors (1904A and 1904B) using respectively the EF1α and the MND promoters to drive gene expression and compared them in cell culture and in a preclinical study. Methods: The second-generation CD19-CAR with T2A-linked tEGFR was expressed by using the EF1α or the MND promoter. The expression levels of tEGFR and CAR were determined by flow cytometry using the biotinylated-Erbitux, and the CD19-Fc protein and the α-FMC63scFv antibody, respectively. The in vitro cytotoxicity assay was performed by co-culture of CAR-T and Calcein-AM-stained K562-CD19. NOD-SCID mice engrafted with Raji-luciferase cells were used as an animal model to validate the activities of 1904A and 1904B CAR-T. A randomized, double-blinded, and dual-arms prospective clinical study was registered (NCT03840317) to compare the safety and efficacy of the treatments for recurrent/refractory (r/r) B-ALL. Subjects were assigned in two groups: those with blasts (by morphology) in bone marrow between 5-20% or above 20%. All subjects were preconditioned with fludarabine and cyclophosphamide before 3x105 CAR-T cells/kg infusion. Results: While tEGFR expression in 1904A and 1904B CAR-T cells was similar (with relative MFIs of 10.47±1.16 and 12.19±1.30 respectively), a significant increase in CAR expression was detected in 1904A compared to 1904B (with relative MFIs of 2.26±0.37 vs 1.24±0.07 and 4.37±0.59 vs 1.13±0.08 as determined by α-FMC63scFv and CD19Fc staining), indicating increased CAR expression on the surface of 1904A CAR-T cells. Importantly, 1904A cells showed greater cytotoxicity and release of cytokines, especially IFNγ and TNFα in cell culture. Both 1904A and 1904B treatments prolonged the median survival time in the animal model. Interestingly, the increase of tumor cells in mice was slower in the group treated with 1904B, suggesting 1904B T cells were more susceptible to the inhibitory effect of the tumor. In the clinical study, only 6 and 8 subjects for 1904A and 1904B respectively completed the trial by the end of July 2019. The median follow-up for 1904A and 1904B were 127 (67-168) and 138.5 (45-179) days, and tumor burdens were 3.06% (0.68%-82.7%) and 4.49% (1.29%-39.0%), respectively. The adverse events of subjects were monitored and recorded according to CTCAE 5.0. No significant difference in disease incidence or severity was detected between the two groups. The CR rate is 83.3% (5/6) for the 1904A group, and 100% (8/8) for the 1904B group. There were 3 (1904A) and 5 (1904B) subjects who bridged to HSCT. All subjects remained in remission with one CD19-negative recurrence in the 1904B group. Conclusion: Using the myeloproliferative sarcoma virus enhancer, we have generated a lentivirus vector, 1904B that can express CAR at a lower level on the cell surface, and investigated 1904B CAR-T functions in cell culture, in a mouse model, and in patients. Lower surface expression of CAR was found with reduced cytotoxicity and lower cytokine release of CAR-T in cell culture, but prolonged T cell activity in a mouse model. Human subjects treated with 1904B CAR-T developed fever later and shorter than 1904A, with slower expansion of CAR-T cells. We suggest that a lower surface expression of CAR molecules can decelerate the kinetics and decrease the intensity of CAR-T activation in vitro and in vivo. Figure Disclosures No relevant conflicts of interest to declare.

A novel isothiocyanate derivative inhibits HIV-1 gene expression and replication by modulating the nuclear matrix associated protein SMAR1

The essential role of SMAR1 in HIV-1 transcription and LTR driven gene expression suggests SMAR1 as an HIV dependency factor (HDF) and a potential anti-HIV therapeutic target. Here, we report for the first time, anti-HIV activity of 8 novel isothiocyanate (ITC) derivatives that differentially stabilise SMAR1. Out of 8 novel ITC derivatives, SCS-OCL-381 was observed to inhibit HIV-1 replication most significantly at the noncytotoxic concentration in reporter T-cell line, CEM-GFP. Further, the highly conserved anti-HIV activity of SCS-OCL-381 is a cell type, virus isolate and viral load independent phenomena and is approximately 3 fold more effective than the representative ITC, Sulforaphane (SFN). Further, SCS-OCL-381 does not hamper the activity of viral enzymes reverse transcriptase, integrase and protease. Mechanistically, SCS-OCL-381 stabilises SMAR1 which, otherwise undergoes proteasomal degradation upon HIV-1 infection in T-cells. This stabilisation results in the recruitment of repressor complex on HIV-1 LTR resulting in repression of LTR mediated transcription and gene expression. These inhibitory consequences were further confirmed by reporter based LTR activity assays in different cell lines. Taken together, these findings highlight the anti-HIV potential of novel ITC derivatives by the stabilisation of SMAR1 and strongly support further in vivo characterisation and potential translational applications of SCS-OCL-381.

Discovery and Characterization of Native Deinococcus radiodurans Promoters for Tunable Gene Expression.

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radioduransIMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.

IS tRNA DRIVING BREAST CANCER PROGRESSION?

This critical research periodical is mainly based on critical review of research article titled ‘Modulated Expression of Specific tRNAs Drives Gene Expression and Cancer Progression published in Cell by Goodarzi et al1. According to Globocan, 2008 report2, breast is among the leading site of new cancer cases and deaths (691,300/268,900) in females of developing countries and second leading site in USA (Globocan, 2012)3. The extensive research is in progress on different aspects of molecular mechanism of driving forces and different treatment modalities to ease this burden. The above mentioned research article is also part of this effort.

Context-dependency of synthetic minimal promoters in driving gene expression: a case study.

SummarySynthetic promoters are considered ideal candidates in driving robust gene expression. Most of the available synthetic promoters are minimal promoters, for which the upstream sequence of the 5′ end of the core region is usually excluded. Although the upstream sequence has been shown to mediate transcription of natural promoters, its impact on synthetic promoters has not been widely studied. Here, a library of chromosomal DNA fragments is randomly fused with the 5′ end of the J23119 synthetic promoter, and the transcriptional performance of the promoter is evaluated through β‐galactosidase assay, fluorescence intensity and chemical biosynthesis. Results show that changes in the upstream sequence can induce significant variation in the promoter strength of up to 5.8‐fold. The effect is independent of the length of the insertions and the number of potential transcription factor binding sites. Several DNA fragments that are able to enhance the transcription of both the natural and the synthetic promoters are identified. This study indicates that the synthetic minimal promoters are susceptible to the surrounding sequence context. Therefore, the upstream sequence should be treated as an indispensable component in the design and application of synthetic promoters, or as an independent genetic part for the fine‐tuning of gene expression.

Drift and Directional Selection Are the Evolutionary Forces Driving Gene Expression Divergence in Eye and Brain Tissue of Heliconius Butterflies.

Investigating gene expression evolution over micro- and macroevolutionary timescales will expand our understanding of the role of gene expression in adaptation and speciation. In this study, we characterized the evolutionary forces acting on gene expression levels in eye and brain tissue of five Heliconius butterflies with divergence times of ∼5-12 MYA. We developed and applied Brownian motion (BM) and Ornstein-Uhlenbeck (OU) models to identify genes whose expression levels are evolving through drift, stabilizing selection, or a lineage-specific shift. We found that 81% of the genes evolve under genetic drift. When testing for branch-specific shifts in gene expression, we detected 368 (16%) shift events. Genes showing a shift toward upregulation have significantly lower gene expression variance than those genes showing a shift leading toward downregulation. We hypothesize that directional selection is acting in shifts causing upregulation, since transcription is costly. We further uncovered through simulations that parameter estimation of OU models is biased when using small phylogenies and only becomes reliable with phylogenies having ≥ 50 taxa. Therefore, we developed a new statistical test based on BM to identify highly conserved genes (i.e., evolving under strong stabilizing selection), which comprised 3% of the orthoclusters. In conclusion, we found that drift is the dominant evolutionary force driving gene expression evolution in eye and brain tissue in Heliconius Nevertheless, the higher proportion of genes evolving under directional than under stabilizing selection might reflect species-specific selective pressures on vision and the brain that are necessary to fulfill species-specific requirements.

Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.

Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell-Specific Gene Promoters.

BackgroundBrain metastases (BrM) develop in 20–40% of cancer patients and represent an unmet clinical need. Limited access of drugs into the brain because of the blood-brain barrier is at least partially responsible for therapeutic failure, necessitating improved drug delivery systems.MethodsGreen fluorescent protein (GFP)-transduced murine and nontransduced human hematopoietic stem cells (HSCs) were administered into mice (n = 10 and 3). The HSC progeny in mouse BrM and in patient-derived BrM tissue (n = 6) was characterized by flow cytometry and immunofluorescence. Promoters driving gene expression, specifically within the BrM-infiltrating HSC progeny, were identified through differential gene-expression analysis and subsequent validation of a series of promoter-green fluorescent protein-reporter constructs in mice (n = 5). One of the promoters was used to deliver tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) to BrM in mice (n = 17/21 for TRAIL vs control group).ResultsHSC progeny (consisting mostly of macrophages) efficiently homed to macrometastases (mean [SD] = 37.6% [7.2%] of all infiltrating cells for murine HSC progeny; 27.9% mean [SD] = 27.9% [4.9%] of infiltrating CD45+ hematopoietic cells for human HSC progeny) and micrometastases in mice (19.3–53.3% of all macrophages for murine HSCs). Macrophages were also abundant in patient-derived BrM tissue (mean [SD] = 8.8% [7.8%]). Collectively, this provided a rationale to optimize the delivery of gene therapy to BrM within myeloid cells. MMP14 promoter emerged as the strongest promoter construct capable of limiting gene expression to BrM-infiltrating myeloid cells in mice. TRAIL delivered under MMP14 promoter statistically significantly prolonged survival in mice (mean [SD] = 19.0 [3.4] vs mean [SD] = 15.0 [2.0] days for TRAIL vs control group; two-sided P = .006), demonstrating therapeutic and translational potential of our approach.ConclusionsOur study establishes HSC gene therapy using a myeloid cell–specific promoter as a new strategy to target BrM. This approach, with strong translational value, has potential to overcome the blood-brain barrier, target micrometastases, and control multifocal lesions.

Signal Transduction in Diffuse Intrinsic Pontine Glioma.

Diffuse intrinsic pontine glioma (DIPG) is an untreatable, heterogeneous high-grade glioma (HGG) of the brainstem. This highly aggressive cancer affects mostly young children and is uniformly fatal. Genomic studies show that DIPG is driven by somatic mutations to histone H3, either H3.1 or H3.3 variants (HIST1H3B/C and H3F3A), altering the epigenetic landscape of primitive oligodendrocyte or astrocyte precursor cells of the pontine region of the brainstem. Lysine-to-methionine point mutations at amino acid 27 (H3K27M) co-occur with alterations in signaling genes, including the receptor tyrosine kinases (PDGFR/KIT/VEGFR/MET/EGFR), activin A receptor (ACVR1), intracellular kinases (PI3K/AKT/mTOR), cyclin-dependent kinases (CDKs1/4/6), transcriptional regulators (MYCN), and tumor suppressors (PTEN/TP53). This cooperation drives gene expression signatures that inhibit cellular differentiation (ID1/2, Hedgehog) and promotes malignant transformation. Unique to DIPG, is the frequency of co-occurring sets of genomic insults. However, mapping of the oncogenic signaling pathways activated in response to recurring mutations is unresolved. Herein, known oncogenic signal pathways activated in response to recurring somatic mutations and gene amplifications in DIPG are reviewed. Additionally, an important role for high-resolution quantitative proteomics/phosphoproteomics in the characterization of signaling cascades are highlighted. These regulate the cell cycle, epigenetics and anti-apoptotic processes, information critical for the development of improved treatment strategies for DIPG.