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Genome-wide association studies for severe malaria (SM) have identified 30 genetic variants mostly located in non-coding regions. Here, we aimed to identify potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium (LD) with the malaria-associated genetic variants. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing five ATP2B4 SNPs in LD with rs10900585. We found significant associations between SM and rs10900585 and our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we demonstrated that both individual SNPs and the combination of SNPs had regulatory effects. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Our data demonstrate that severe malaria-associated genetic variants alter the expression of ATP2B4 encoding a plasma membrane calcium-transporting ATPase 4 (PMCA4) expressed on red blood cells. Altering the activity of this regulatory element affects the risk of SM, likely through calcium concentration effect on parasitaemia. [ABSTRACT FROM AUTHOR]

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP‐like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high‐resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M−1 cm−1) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors. PDB Code(s): 7SFA and 7SF9; [ABSTRACT FROM AUTHOR]

Abstract Background In dairy cows circulating non-esterified fatty acids (NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors (PPAR). PPAR are activated by fatty acids (FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows. Methods The dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300e digital dispenser and luciferase reporter in several bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase (LPL), and palmitic acid (C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors. Results NEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum (R2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation. Conclusion Our results support dose-dependent activation of PPAR by circulating NEFA in bovine, specifically δ and γ isotypes. Data also support the possibility of increasing PPAR activation by dietary FA; however, this nutrigenomics approach maybe only effective in pre-partum but not post-partum cows.

Background: In dairy cows circulating non-esterified fatty acids (NEFA) increase early post-partum while liver and other tissues undergo adaptation to greater lipid metabolism, mainly regulated by peroxisome proliferator-activated receptors (PPAR). PPAR are activated by fatty acids (FA), but it remains to be demonstrated that circulating NEFA or dietary FA activate bovine PPAR. We hypothesized that circulating NEFA and dietary FA activate PPAR in dairy cows. Methods: The dose-response activation of PPAR by NEFA or dietary FA was assessed using HP300e digital dispenser and luciferase reporter in several bovine cell types. Cells were treated with blood plasma isolated from Jersey cows before and after parturition, NEFA isolated from the blood plasma, FA released from lipoproteins using milk lipoprotein lipase (LPL), and palmitic acid (C16:0). Effect on each PPAR isotype was assessed using specific synthetic inhibitors. Results: NEFA isolated from blood serum activate PPAR linearly up to ~ 4-fold at 400 μmol/L in MAC-T cells but had cytotoxic effect. Addition of albumin to the culture media decreases cytotoxic effects of NEFA but also PPAR activation by ~ 2-fold. Treating cells with serum from peripartum cows reveals that much of the PPAR activation can be explained by the amount of NEFA in the serum (R2 = 0.91) and that the response to serum NEFA follows a quadratic tendency, with peak activation around 1.4 mmol/L. Analysis of PPAR activation by serum in MAC-T, BFH-12 and BPAEC cells revealed that most of the activation is explained by the activity of PPARδ and PPARγ, but not PPARα. Palmitic acid activated PPAR when added in culture media or blood serum but the activation was limited to PPARδ and PPARα and the response was nil in serum from post-partum cows. The addition of LPL to the serum increased > 1.5-fold PPAR activation. Conclusion: Our results support dose-dependent activation of PPAR by circulating NEFA in bovine, specifically δ and γ isotypes. Data also support the possibility of increasing PPAR activation by dietary FA; however, this nutrigenomics approach maybe only effective in pre-partum but not post-partum cows. [ABSTRACT FROM AUTHOR]

Genetic constructs with promoters fused to reporter genes for simultaneous monitoring of cellular events have been the focus of attention in recent years. Adenoviral vectors, which have distinctive characteristics, have been used to monitor the differentiation of stem cells in vitro. In the present study, a modified adenoviral vector was constructed, containing a mouse, rat, and human general albumin promoter sequence fused to a ZsGreen reporter gene, and evaluated its efficiency in different cell types. Two hepatocyte cell lines (Hepa1-6 and HepG2), rat primary hepatocytes, rat bone marrow mesenchymal stem cells (BM-MSCs) and rat BM-MSCs-derived hepatocyte-like cells were transduced with this vector, and the transfection efficiency and functional capabilities of the promoter were evaluated by fluorescent microscopy. The results demonstrated efficient expression of ZsGreen in Hepa1-6 cells, HepG2 cells, rat primary hepatocytes, and rat BM-MSCs-derived hepatocyte-like cells, but not in rat BM-MSCs. In conclusion, the current study demonstrates a simple, high-efficiency, general tool for real-time monitoring of the differentiation status of hepatocytes from stem cells in mice, rats, and humans. This tool may be useful for evaluating different protocols to generate functional hepatocytes from stem cells in multiple species. [ABSTRACT FROM AUTHOR]

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.

RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D), previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP) and firefly luciferase (repYFV-17D-Luc) reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.O replicon de RNA derivado do genoma de Flavivirus é uma ferramenta valiosa para o estudo de replicação viral independente da montagem e da maturação do virion, além de possuir um grande potencial para expressão de genes heterólogos. Neste estudo nós descrevemos a construção de replicons subgenômicos do vírus da febre amarela utilizando a técnica de recombinação homóloga em levedura. O plasmídeo contendo o replicon do vírus febre amarela cepa 17D (pBSC-repYFV-17D), caracterizado anteriormente, foi manipulado para a expressão heteróloga dos genes repórteres green fluorescent protein (repYFV-17D-GFP) e firelly luciferase (repYFV-17D-Luc). Ambos os replicons foram construídos por recombinação homóloga entre o vetor pBSC-repYFV-17D linearizado e o produto de PCR contendo 25 nucleotídeos terminais homólogos incorporados aos oligonucleotídeos iniciadores. A organização genômica dos construídos é semelhante ao repYFV-17D, com inserção de um gene repórter entre os restantes 63 nucleotídeos N-terminais da proteína do capsídeo e 72 nucleotídeos C-terminais da proteína E. Os replicons repYFV-17D-GFP e repYFV-17D-Luc mostraram uma eficiente replicação e expressão dos genes repórteres. A técnica de recombinação homóloga em levedura usada neste estudo demonstrou ser aplicável à manipulação do genoma do vírus da febre amarela para a construção de replicons subgenômicos.

Purpose To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [13C]urea. Methods Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [13C]urea, a spin echo sequence was used to measure urea concentration, T1, and diffusion in control and UTB-expressing tissue. Results The apparent diffusion coefficient of hyperpolarized urea was 21% lower in tissue expressing UTB, in comparison with control tissue ( P < 0.05, 1-tailed t-test, n = 6 in each group). No difference in water apparent diffusion coefficient or cellularity between these tissues was found, indicating that they were otherwise similar in composition. Conclusion Expression of UTB, by mediating cell uptake of urea, lowers the apparent diffusion coefficient of hyperpolarized 13C urea in tissue and thus the transporter has the potential to be used as a magnetic resonance-based gene reporter in vivo. Magn Reson Med 73:1401-1406, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The main objective of cell therapy is the regeneration of damaged tissues. To distinguish graft from host tissue by magnetic resonance imaging (MRI), a paramagnetic label must be introduced to cells prior to transplantation. The paramagnetic label can be either exogenous iron oxide nanoparticles or a genetic overexpression of ferritin, an endogenous iron storage protein. The purpose of this work was to compare the efficacy of these 2 methods for MRI evaluation of engrafted cell survival in the infarcted mouse heart. Mouse skeletal myoblasts were labeled either by cocultivation with iron oxide particles or by engineering them to overexpress ferritin. Along with live cell transplantation, 2 other groups of mice were injected with dead-labeled cells. Both particle-labeled and ferritin-tagged grafts were detected as areas of MRI signal hypointensity in the left ventricle of the mouse heart using T2*-weighted sequences, although the signal attenuation decreased with ferritin tagging. Importantly, live cells could not be distinguished from dead cells when labeled with iron oxide particles, whereas the ferritin tagging was detected only in live grafts, thereby allowing identification of viable grafts using MRI. Thus, iron oxide particles can provide information about initial cell injection success but cannot assess graft viability. On the other hand, genetically based cell tagging, such as ferritin overexpression, despite having lower signal intensity in comparison with iron oxide particles, is able to identify live transplanted cells. [ABSTRACT FROM AUTHOR]

Compelling evidence has shown that the effects of antidepressants, increasing extracellular serotonin and noradrenaline as a primary mechanism of action, involve neuroplastic and neurotrophic mechanisms. Brain-derived neurotrophic factor (BDNF) has been shown to play a key role in neuroplasticity and synaptic function, as well as in the pathophysiology of neuropsychiatric disorders and the mechanism of action of antidepressants. The expression of BDNF is mediated by the transcription of different mRNAs derived by the splicing of one of the eight 5′ non-coding exons with the 3′ coding exon (in rats). The transcription of each non-coding exon is driven by unique and different promoters. We generated a gene reporter system based on hippocampal and cortical neuronal cultures, in which the transcription of luciferase is regulated by BDNF promoters I, II, IV or by cAMP response element (CRE), to investigate the activation of selected promoters induced by monoaminergic antidepressants and by serotonin or noradrenaline agonists. We found that incubation with fluoxetine or reboxetine failed to induce any activation of BDNF promoters or CRE. On the other hand, the incubation of cultures with selective agonists of serotonin or noradrenaline receptors induced a specific and distinct profile of activation of BDNF promoters I, II, IV and CRE, suggesting that the monoaminergic input, absent in dissociated cultures, is essential for the modulation of BDNF expression. In summary, we applied a rapidly detectable and highly sensitive reporter gene assay to characterize the selective activation profile of BDNF and CRE promoters, through specific and different pharmacological stimuli. [ABSTRACT FROM PUBLISHER]

Saccharomyces cerevisiae is able to sense and respond to environmental changes such as the availability of carbon sources. In a previous work, we showed that the expression of the Pre S2- S gene of HBV in yeast was negatively regulated at the translational level dependent of glucose. In this study, we show that the S m RNA is detected in the polysomes indicating its active translation, while the Pre S2- S m RNA was mainly found in monosomes. Moreover, we used the gene reporter assay based on Zeocin resistance, to better characterize the Pre S2 region responsible for this control. Two chimeric genes composed of the N- and C-terminal part of the Pre S2 fused to the Sh-bleomycin gene conferring the resistance to Zeocin were expressed in yeast. We found that the strain expressing the N-terminal part of the Pre S2 was sensitive to Zeocin on rich medium with 2% glucose. In contrast, the strain harbouring the C-terminal part of the Pre S2 fused to the Sh-bleomycin grew on Zeocin, indicating that the Sh-bleomycin m RNA is efficiently translated, subsequently conferring resistance to Zeocin. Our data suggest the establishment of a translational control via the N-terminal part of the Pre S2 mediated by the presence of 2% glucose in the media. [ABSTRACT FROM AUTHOR]

The phenylacetic acid (PA) degradative pathway is the central pathway by which complex aromatic compounds (e.g. styrene) get degraded. Upper pathways for different aromatic compounds converge at common intermediate phenylacetyl-CoA, which is then metabolized to succinyl-CoA and acetyl-CoA. We previously made a link in Burkholderia cenocepacia between phenylacetic acid (PA) degradation and virulence by showing that insertional mutagenesis of paaA and paaE results in PA-conditional growth and an attenuated killing phenotype in the Caenorhabditis elegans model of infection. Insertional mutagenesis of paaK1, which encodes a phenylacetyl-CoA ligase, did not result in a PA-conditional growth probably due to the presence of a second similar gene (paaK2) encoding another phenylacetyl-CoA ligase (PaaK2). Recently published crystallographic and enzyme kinetics studies comparing the two ligases show that PaaK1 is better able to bind hydroxylated PA derived molecules such as 3-hydroxyphenylacetic (3-OHPA) acid and 4-hydroxyphenylacetic acid (4-OHPA) due to its having a larger binding pocket than PaaK2. However, the biological significance of this finding remains unanswered. In this study, we demonstrate that 3-OHPA but not 4-OHPA, induces the phenylacetic acid degradative pathway in Burkholderia cenocepacia K56-2. Using reporter activity assays, we show paaK1-dependent induction of the PA degradative pathway when 3-OHPA is added to the culture medium. To shed light on a possible relevance of this finding to pathogenicity, we compared the pathogenicity of the paaK1 deletion mutant with that of the wild type in C. elegans. Results show that the loss of PaaK1 function does not equal to a loss of pathogenicity. Taken together our results suggest that 3-OHPA but not 4-OHPA may be degraded through the PA degradative pathway but that 3-OHPA-related intermediates may not be relevant to pathogenesis as previously thought.

Transformação genética estável de plantas é geralmente um processo prolongado, envolvendo em muitos casos mais de um ano de regeneração, propagação e seleção de transgénicos. Análises de expressão transiente são úteis para economizar tempo em estudos de função de genes, particularmente em genes que expressam em fruto. Sistema de expressão transiente de um gene repórter em frutos de banana foi então desenvolvido. Em frutos maduros, Agrobacterium tumefaciens, cujo plasmídio contem o gene de gusintron sob o controle do promotor de CaMV 35S, foi infiltrada nos frutos por meio de uma seringa. Em frutos imaturos, as Agrobacteria foram infiltradas por vácuo nos frutos fatiados e foram co-cultivados. Ensaio histoquímico de GUS foi realizado a 3 dias depois da infiltração ou co-cultivo. A manipulação dos frutos maduros na análise histoquímica era difícil e baixa expressão de GUS foi obtida. Por outro lado, os frutos imaturos fatiados eram fáceis de manipular e alto nível de expressão foi observado. O sistema de expressão transiente desenvolvido neste estudo pode ser útil para análises rotineiras de validação de função de genes em frutos.

Abstract: Background: Adipose tissue-derived progenitor cells (ATDPCs) isolated from human cardiac adipose tissue are useful for cardiac regeneration in rodent models. These cells do not express cardiac troponin I (cTnI) and only express low levels of PECAM-1 when cultured under standard conditions. The purpose of the present study was to evaluate changes in cTnI and PECAM-1 gene expression in cardiac ATDPCs following their delivery through a fibrin patch to a murine model of myocardial infarction using a non-invasive bioluminescence imaging procedure. Methods and results: Cardiac and subcutaneous ATDPCs were doubly transduced with lentiviral vectors for the expression of chimerical bioluminescent–fluorescent reporters driven by constitutively active and tissue-specific promoters (cardiac and endothelial for cTnI and PECAM-1, respectively). Labeled cells mixed with fibrin were applied as a 3-D fibrin patch over the infarcted tissue. Both cell types exhibited de novo expression of cTnI, though the levels were remarkably higher in cardiac ATDPCs. Endothelial differentiation was similar in both ATDPCs, though cardiac cells induced vascularization more effectively. The imaging results were corroborated by standard techniques, validating the use of bioluminescence imaging for in vivo analysis of tissue repair strategies. Accordingly, ATDPC treatment translated into detectable functional and morphological improvements in heart function. Conclusions: Both ATDPCs differentiate to the endothelial lineage at a similar level, cardiac ATDPCs differentiated more readily to the cardiomyogenic lineage than subcutaneous ATDPCs. Non-invasive bioluminescence imaging was a useful tool for real time monitoring of gene expression changes in implanted ATDPCs that could facilitate the development of procedures for tissue repair. [Copyright &y& Elsevier]

Purpose: Wide-field surgical excision reduces the chance of residual disease, but can also lead to disfigurement and devastating morbidities when resection is close to critical structures. We hypothesize that near-infrared fluorescence (NIRF) imaging can enable accurate detection of tumor margins for image-guided resection. Experimental Design: An orthotopic model of human prostate cancer (PCa) was used to assess primary tumor margins using a NIRF-labeled antibody against epithelial cell adhesion molecule (EpCAM). PCa cells stably expressing far red fluorescent gene reporter, iRFP, enabled colocalization with NIRF signals for direct assessment of tumor margins. Results: Using receiver operating characteristic analysis, far red fluorescence was validated against standard pathology of primary and metastatic lesions with >96 % accuracy. Primary tumor margins were more accurately detected by quantitative NIRF imaging using the EpCAM-targeting antibody as compared to a NIRF-labeled isotype control antibody. Conclusions: NIRF molecular imaging may enable real-time and accurate assessment of tumor margins. [ABSTRACT FROM AUTHOR]

Abstract: Glucose transporter GLUT4 protein, codified by Slc2a4 gene plays a key role in glycemic homeostasis. Insulin resistance, as in obesity, has been associated to inflammatory state, in which decreased GLUT4 is a feature. Inflammatory NF-κB transcriptional factor has been proposed as a repressor of Slc2a4; although, the binding site(s) in Slc2a4 promoter and the direct repressor effect have never been reported yet. A motif-based sequence analysis of mouse Slc2a4 promoter revealed two putative κB sites located inside −83/−62 and −134/−113bp. Eletrophoretic mobility assay showed that p50 and p65 NF-κB subunits bind to both putative κB sites. Chromatin immunoprecipitation assay using genomic DNA from adipocytes confirmed p50- and p65-binding to Slc2a4 promoter. Moreover, transfection experiments revealed that NF-κB binds to the −134/−113bp region of the mouse Slc2a4 gene promoter, inhibiting the Slc2a4 gene transcription. The current findings demonstrate the existence of two κB sites in Slc2a4 gene promote, and that NF-κB has a direct repressor effect upon the Slc2a4 gene, providing an important link between insulin resistance and inflammation. [Copyright &y& Elsevier]

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Abstract: Green fluorescent protein (GFP) is a widely utilized molecular marker of gene expression. However, its use in in vivo imaging has been restricted to transparent tissue mainly due to the tissue penetrance limitation of optical imaging. Here, we report a novel approach to detect GFP with Magnetization transfer contrast (MTC) magnetic resonance imaging (MRI). MTC is an MRI methodology currently utilized to detect macromolecule changes such as decrease in myelin and increase in collagen content. We employed MTC MRI imaging to detect GFP both in vitro and in in vivo mouse models. We demonstrated that our approach produces values that are protein specific, and concentration dependent. This approach provides a flexible, non-invasive in vivo molecular MRI imaging strategy that is dependent upon the presence and concentration of the GFP reporter. [Copyright &y& Elsevier]

Abstract: The CB1 cannabinoid receptor shows complex interactions with intracellular signalling partners, and responses to cannabinoid ligands are likely to be influenced by concomitant inputs modifying the overall tone of signalling cascades. This appears even more relevant as we previously evidenced opposite regulations of tyrosine hydroxylase (TH) expression by the two common cannabinoid agonists HU 210 and CP 55,940. Therefore, we studied the consequences of manipulating adenylyl cyclase activity with forskolin on the regulation of TH gene transcription in neuroblastoma cells (N1E-115). Reporter gene experiments performed with the luciferase sequence cloned under the control of modified fragments of the TH gene promoter revealed that the AP-1 consensus sequence is essential for cannabinoid-mediated regulation of TH expression. Consistently, inhibition of PKC totally blocked the responses mediated by both HU 210 and CP 55,940. In addition, forskolin which boosts adenylyl cyclase activity remarkably modified the responses to the cannabinoid agonists. Thus, in these conditions, both agonists efficiently reduced TH gene promoter activity, a response requiring functional PKA/CRE-dependent signallings. Finally, the modulations of the promoter were inhibited in pertussis toxin treated cells, suggesting that responses to both agonists are mediated through Gi/o-dependent mechanisms. Emphasising on the importance of functional selectivity at GPCRs, these data demonstrate that the concomitant activation of adenylyl cyclase by forskolin strongly influences the biochemical responses triggered by distinct cannabinoid agonists. Together our results suggest that the physiological modulation of TH expression by cannabinoid agonists in dopaminergic neurons would be influenced by additional endogenous inputs. [Copyright &y& Elsevier]

Abstract: Biofilms possess spatially and temporally varying metabolite concentration profiles at the macroscopic and microscopic scales. This results in varying growth environments that may ultimately drive species diversity, determine biofilm structure and the spatial distribution of the community members. Using non-invasive nuclear magnetic resonance (NMR) microscopic imaging/spectroscopy and confocal imaging, we investigated the kinetics and stratification of anaerobic metabolism within live biofilms of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1. Biofilms were pre-grown using a defined minimal medium in a constant-depth film bioreactor and subsequently transferred to an in-magnet sample chamber under laminar flow for NMR measurements. Biofilms generated in this manner were subjected to changing substrate/electron acceptor combinations (fumarate, dimethyl sulfoxide, and nitrate) and the metabolic responses measured. Localized NMR spectroscopy was used to non-invasively measure hydrogen-containing metabolites at high temporal resolution (4.5 min) under O2-limited conditions. Reduction of electron acceptor under anaerobic conditions was immediately observed upon switching feed solutions indicating that no gene induction (transcriptional response) was needed for MR-1 to switch metabolism from O2 to fumarate, dimethyl sulfoxide or nitrate. In parallel experiments, confocal microscopy was used with constitutively expressed fluorescent reporters to independently investigate changes in population response to the availability of electron acceptor and to probe metabolic competition under O2-limited conditions. A clearer understanding of the metabolic diversity and plasticity of the biofilm mode of growth as well as how these factors relate to environmental fitness is made possible through the use of non-invasive and non-destructive techniques such as described herein. [Copyright &y& Elsevier]

Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding Β-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing Β-galactosidase (Β-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl Β-D-galactopyranoside (OFPNPG) is readily hydrolyzed by Β-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach. [ABSTRACT FROM AUTHOR]

The development of a whole-cell fluorescence-based biosensor for nitrate is reported. The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon (Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP). Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence. The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration. The developed bioassay has established that E. coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine-N-oxide and fumerate, and azide, an inhibitor of redox-active proteins. [Copyright &y& Elsevier]

A continuous culture technique utilising a variant of Staphylococcus aureus 8325-4 containing transcriptional gene fusions was used to investigate the relationships between cell density (OD600), steady-state specific growth rate (μ) and expression of both agr (accessory gene regulator) and tst (toxic shock syndrome toxin-1). The expression of these genes was assessed by two single-copy independently arranged chromosomal-based reporter systems, β-galactosidase agr–P3 promoter fusion and a lux–tst promoter fusion. Cell density and specific agr expression were found to be positively correlated. In the model, the minimum cell density predicted to promote specific agr expression was an OD600 of 0.14, equivalent to 1.2×108 CFU ml−1. No direct relationship between cell density and specific tst expression was detected. Specific expressions of agr and tst were not correlated with specific growth rate and there appeared to be no direct link between agr and tst specific expression. The results support the hypothesis that agr is a functional unit of quorum sensing and that the amount of specific expression of tst is modulated independently of agr. [Copyright &y& Elsevier]

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and E c 14,300 M -1  cm -1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors., (© 2021 The Protein Society.)

Abstract: Reporter gene assays are important tools for evaluating gene expression. A frequently used assay measures the activity of β-galactosidase (β-gal) expressed from lacZ in plasmid or genomic constructions. Such constructions are often used to interrogate the ability of DNA (query DNA), potentially encoding a transcription factor, to regulate in trans the expression of a promoter fused to the reporter lacZ. Query DNA is frequently inserted into a second plasmid within the α-subunit of β-gal, interrupting its function. However, this plasmid can induce up-expression of β-gal even when void of query DNA, leading to confusion between artifact and authentic regulation. [Copyright &y& Elsevier]

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

Genetic constructs with promoters fused to reporter genes for simultaneous monitoring of cellular events have been the focus of attention in recent years. Adenoviral vectors, which have distinctive characteristics, have been used to monitor the differentiation of stem cells in vitro . In the present study, a modified adenoviral vector was constructed, containing a mouse, rat, and human general albumin promoter sequence fused to a ZsGreen reporter gene, and evaluated its efficiency in different cell types. Two hepatocyte cell lines (Hepa1-6 and HepG2), rat primary hepatocytes, rat bone marrow mesenchymal stem cells (BM-MSCs) and rat BM-MSCs-derived hepatocyte-like cells were transduced with this vector, and the transfection efficiency and functional capabilities of the promoter were evaluated by fluorescent microscopy. The results demonstrated efficient expression of ZsGreen in Hepa1-6 cells, HepG2 cells, rat primary hepatocytes, and rat BM-MSCs-derived hepatocyte-like cells, but not in rat BM-MSCs. In conclusion, the current study demonstrates a simple, high-efficiency, general tool for real-time monitoring of the differentiation status of hepatocytes from stem cells in mice, rats, and humans. This tool may be useful for evaluating different protocols to generate functional hepatocytes from stem cells in multiple species.

Genetic reporters have become invaluable tools for indirectly monitoring promoter activities. The quantitative measurement of promoter activities using reporter gene systems is fundamental for pharmaceutical, biomedical, and molecular biology research. Genetic reporters are used not only for measuring promoter activities but also for understanding the mechanisms controlling gene transcription and in the identification, and characterization of cis-acting regulatory elements. Fluorescent reporter proteins including enhanced green fluorescent protein (EGFP ) are reliable for monitoring quantitative underlying differences in promoter activities. The emitted fluorescence intensity of the expressed reporter is measured at the single-cell level by flow cytometry and represents a readout for the promoter activities. In this chapter, the protocol for measurement and analyzing of transfected cells expressing the reporter gene EGFP is thoroughly described and fully illustrated.

Purpose: To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [(13) C]urea., Methods: Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [(13) C]urea, a spin echo sequence was used to measure urea concentration, T1 , and diffusion in control and UTB-expressing tissue., Results: The apparent diffusion coefficient of hyperpolarized urea was 21% lower in tissue expressing UTB, in comparison with control tissue (P < 0.05, 1-tailed t-test, n = 6 in each group). No difference in water apparent diffusion coefficient or cellularity between these tissues was found, indicating that they were otherwise similar in composition., Conclusion: Expression of UTB, by mediating cell uptake of urea, lowers the apparent diffusion coefficient of hyperpolarized (13) C urea in tissue and thus the transporter has the potential to be used as a magnetic resonance-based gene reporter in vivo. Magn Reson Med 73:1401-1406, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

The phenylacetic acid (PA) degradative pathway is the central pathway by which various aromatic compounds (e.g., styrene) are degraded. Upper pathways for different aromatic compounds converge at common intermediate phenylacetyl-CoA (PA-CoA), which is then metabolized to succinyl-CoA and acetyl-CoA. We previously made a link in Burkholderia cenocepacia between PA degradation and virulence by showing that insertional mutagenesis of paaA and paaE genes, that encode part of a multicomponent oxidase of PA-CoA, results in PA-conditional growth and an attenuated killing phenotype in the Caenorhabditis elegans model of infection. However, insertional mutagenesis of paaK1, which encodes a phenylacetate-CoA ligase, did not result in a PA-conditional growth probably due to the presence of a putative paralog gene paaK2. Recently published crystallographic and enzyme kinetics data comparing the two PaaK ligases showed that PaaK1 is more active than PaaK2 and that the larger binding pocket of PaaK1 can accommodate hydroxylated PA derived molecules such as 3-hydroxyphenylacetic (3-OHPA) acid and 4-hydroxyphenylacetic acid (4-OHPA). The higher activity and broader substrate specificity suggested a more active role in pathogenesis. In this work, we aimed to determine the relevance of PaaK1 activity to the killing ability of B. cenocepacia to C. elegans. Using reporter activity assays, we demonstrate that 3-OHPA activated PA degradation gene promoters of Burkholderia cenocepacia K56-2 in a paaK1-dependent manner, while 4-OHPA had no effect. We compared the pathogenicity of a paaK1 deletion mutant with that of the wild type in C. elegans and observed no differences in the killing ability of the strains. Taken together, these studies suggest that 3-OHPA, but not 4-OHPA, can induce the PA pathway and that this induction is dependent on the paaK1 gene. However, the more active PaaK1 does not play a distinct role in pathogenesis of B. cenocepacia as previously suggested.