Day: September 24, 2021

How the Sapphire Scanner is Used to Better Grocery Store Tomatoes

Categories
Imaging Western Blotting

Few things are more disappointing than a tasteless, mealy grocery store tomato. These bland fruits are pale imitations of the vine-ripened tomatoes available from the garden at the end of summer. Tomatoes are perishable, and providing ripe, high-quality tomatoes that maintain their texture and nutritional content is a challenge for commercial growers.

A recent publication by Tsafouros et al from the Institute of Olive Tree in Greece provides a window into the intense, ongoing research aimed at understanding how and why tomatoes and other fruits ripen under various conditions. Such studies could improve the postharvest shelf-life and the quality of commercially grown tomatoes.

Growing the tomatoes used in this study

For this study, tomatoes were grown in a greenhouse and either picked at “commercial maturity” (when the tomato is just turning color) or left on the plant to mature for an additional week. The harvested tomatoes were stored at 5, 10, or 25 °C and after 7 days were compared to each other and to tomatoes left to ripen on the vine.

Tomatoes are typically picked before ripening and stored and transported at low temperatures in an attempt to increase the shelf-life. Tsafouros et al examined the effect on ripening of storing picked tomatoes at various temperatures for a week. The authors also characterized in detail the effect of storage temperature on the metabolism of polyamines, compounds known to play a role in fruit ripening and the content of which are known to be associated with tomato quality and shelf life.

The role of temperature in mediating postharvest polyamine homeostasis in tomato fruit
Chemiluminescent Western blot showing the role of temperature in mediating postharvest polyamine homeostasis in tomato fruit

How the research was conducted

The authors carried out an exhaustive biochemical and molecular biological characterization of polyamine metabolism in the tomatoes. They assessed the total content of a variety of polyamines, the activity of the enzyme responsible for breaking down polyamines, the expression of all 23 genes encoding factors known to be involved in polyamine metabolism, the levels of the proteins involved in polyamine synthesis, and the levels of hydrogen peroxide, a biproduct of amine oxidases acting on polyamines. Protein levels were measured by chemiluminescent Western blots imaged using the Sapphire Biomolecular Imager.

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

The new Sapphire FL images phosphor screens with high sensitivity. With customizable and user-changeable laser and filter modules, it easily adapts to a lab’s changing needs and advancing research.

Learn more about the Sapphire imager and how it can support your research by requesting a quote.

The findings

Their results demonstrate that cold storage alters polyamine metabolism, and support storage of tomatoes at 10°C after picking at commercial maturity. Lower temperatures appeared to induce a stress response, perhaps to protect against chilling injuries, while higher temperatures were associated with lower polyamine levels and lower quality fruit.

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Deciphering Lipid Metabolism in Borrelia burgdorferito Better Understand Lyme Disease

Categories
Antibodies Protein Assays

Lyme disease is the most common tick-born disease in the United States, with almost half a million cases diagnosed every year. Lyme disease is caused by the bacterium Borrelia burgdorferi (Bb), which is transmitted to humans when they are bitten by an infected tick.

In recent work, Hove et al from Colorado State University in Denver, USA, identified a potential galactosyltransferase, BB0572, in the Bb genome by searching the Bb genome for sequences similar to a known glycosyltransferase from a related bacterium.

The group demonstrated that the gene encoding BB0572 was actively expressed by the Bb bacterium. To prove BB0572 has galactosyltransferase activity, the group carried out assays with 14C-labeled cholesterol and extracts of Bb bacteria and of E coli engineered to overexpress the gene for the BB0572 protein. The products of the assay were separated using thin-layer chromatography (TLC), and the radioactive products detected on the TLC plates using a Sapphire Biomolecular Imager.

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

The findings

Many people with Lyme disease have antibodies to a cholesterol-based glycolipid, ACGal (produced by Bb), leading some scientists to propose ACGal as a potential Lyme disease vaccine candidate. How the bacterium produces ACGal, and the related CGal, has been unclear since Bb cannot make its own cholesterol and must modify cholesterol from the host.

Fig 5A. TLC of lipids from cell free assays for the enzymatic incorporation of [26- 14C] cholesterol into cholesteryl-β-D-galacto-pyranoside (CGal).The formation of cholesteryl-β-D-galacto-pyranoside (CGal) (arrow) in whole cell lysates of Bb and recombinant E. coli expressing WT bb0572 (lane 1 and 2). As expected, CGal was not formed by the whole cell lysate of E. coli expressing mutant bb0572 ΔCFF/I/DGD (lane 3). Lane 4 is the empty expression vector control, and lane 6 and 7 are from boiled whole cell lysates of Bb and recombinant E. coli expressing WT bb0572, respectively. Note lane 5 was not used.

The results indicate CGal can be synthesized by BB0572. ACGal was not detected, possibly because the acyl donor was missing from the reaction. The identification of BB0572 as the enzyme likely responsible for the synthesis of CGal and ACGal opens up new areas of research including identifying how ACGal is synthesized from CGal and studying the importance of these lipids to both Bb physiology and the development of Lyme disease.

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Background on Borrelia burgdorferi

Bb does not produce any toxins, and the pathogenesis of Lyme disease in humans appears to be due to a strong immune response to the bacterium itself. The resulting long-lasting inflammatory response can damage several different organs including the brain, peripheral and central nervous systems, heart, and joints.

When Bb infection is recognized immediately, usually by a characteristic bullseye shaped rash around the site of the tick bite, Lyme disease can usually be cured by treatment with antibiotics. However, 20% to 30% of people with Lyme disease will not have the rash, and many will not know they are infected in time to be cured. This has led to a great interest in developing a vaccine to prevent Lyme disease.

In addition to phosphor imaging to detect radiolabeled targets, the Sapphire provides chemiluminescence, multi-channel fluorescence, NIR fluorescence, densitometry, and white light imaging of blots, gels, tissues, and more.

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Pathological Signature of T Helper 17 Cells Associated with Endometriosis Identified

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Uncategorized

Nearly 10% of reproductive age women are affected by endometriosis globally, a condition in which the uterine tissue grows ectopically, resulting in pelvic pain and infertility. The peritoneal fluid (PF) surrounding ectopic endometrial tissue contains T helper 17 (Th17) cells. The number of Th17 cells in PF increases with disease severity; however, their function in endometriosis is unknown.

Recently, Jiang et al. used flow cytometry to detect and sort subpopulations of Th17 cells from the PF of patients with moderate to severe endometriosis. The researchers characterized several subsets of Th17 cells that could ultimately be defined by the presence or absence of four chemokine receptors, CCR4, CCR6, IL-17RE, and CD27. The presence of IL-17RE in particular correlated with an increase in Th17 cells in G1 and S-G2/M phase, suggesting increased cell proliferation. RNA-seq was used to further characterize this Th17 cellular subset.

The authors found an increase in oxidative phosphorylation and electron transport chain-related gene expression, which was validated with the Azure Cielo 3 and SYBR Green, thus identifying a pathological signature of a subset of Th17 cells associated with the disease. Additional experiments demonstrated an increase in adenosine triphosphate (ATP) production and reactive oxygen species (ROS), consistent with a role in mitochondrial respiration.

This work highlights the power of using gene expression analysis to characterize novel cellular subtypes relevant to disease. Next steps include determining whether the increase in ATP and ROS levels is a cause or effect of severe endometriosis. Interestingly, in mouse models of autoimmune kidney disease, decreased IL-17RE expression reduced the overall amount of Th17 cells, thus indicating a possible therapeutic target.

If you’re looking to generate reliable gene expression data, learn more about the Azure Cielo 3 used in this study or contact us at info@azurebiosystems.com.

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Regulation of Gene Expression by Enhancer RNAs

Categories
Imaging Quantification Transfers Western Blotting

The regulation of gene expression is a complicated affair. A vast array of control mechanisms exist that can adjust the levels of gene expression products to match the needs of the cell. Messenger RNA (mRNA) can be processed to alter its stability, protein translation from mRNA can be controlled, and the stability and/or activity of the protein can be altered via post-translational modifications. The first point of control of gene expression is the initiation of gene transcription.

Setten et al studied an eRNA transcribed from an enhancer near the gene encoding a transcription factor called CEBPA (CCAAT enhancer-binding protein alpha). CEBPA is a transcription factor involved in many processes, including cell cycle inhibition and tumorigenesis, and expressed in specific cell lineage. It also plays a role in maintaining cell identity. To study CEBPA protein levels, the authors carried out a quantitative near-infrared fluorescent Western blot imaged on an Azure Sapphire™ Biomolecular Imager (Figure 7). To quantify changes in protein level between samples, the fluorescent signal from the Western blot was normalized to the total protein loaded, as was visualized on the Sapphire.

Figure 7 from Setten RL, Chomchan P, Epps EW, et al. (2021) CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA. Licensed under CC BY 4.0. Quantitative fluorescent Western blot showing levels of CEBPA isoforms detected with a goat–anti rabbit 800 secondary antibody from Azure Biosystems (red) and a NIR protein ladder (blue) (panel A). Before blocking the membrane was stained with a NIR fluorescent total protein stain and an image acquired for total protein normalization (panel B).​

The findings

The researchers set out to determine whether an eRNA transcribed from an enhancer 9kb downstream from the transcription start site of the human CEBPA gene was involved in regulating CEBPA expression. They called this eRNA CRED9. The authors found that levels of CEBPA mRNA and the CRED9 eRNA were correlated across several different cell lines; when CRED9 was high, CEBPA mRNA was also high. They then knocked down CRED9 in a cell line and found that when CRED9 levels were reduced, CEBPA mRNA and CEBPA protein levels were also reduced.

Finally, knockdown of CRED9 reduced the amount of a histone H3K27ac bound to the enhancer, indicating that the activity of the enhancer region was reduced. These results lead the authors to propose that CRED9 and other eRNAs may have an active role in enhancer function and gene regulation.

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Requirements of transcription initiation

Transcription initiation has several requirements. The chromatin structure must open to make the gene accessible to the transcriptional machinery. In eukaryotic cells, the promoter sequence of the gene must be bound by transcription factors that direct RNA polymerase to the gene to begin transcription. Transcription initiation is made more likely by the binding of activator proteins to other DNA regions near the promoter called enhancers, which can be located up- or downstream of the transcription start site.

Genome-wide sequencing experiments have revealed that RNA molecules are transcribed from many enhancer regions, indicating the enhancer regions may not merely be binding sites for activator proteins. These enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) and are not translated into proteins. It is possible that eRNAs may simply be the result of non-specific transcription by RNA polymerase and serve as a sign that chromatin is open and accessible to RNA polymerase in a region or DNA. Alternatively, there is evidence some eRNAs may serve an active role in regulating gene expression by themselves binding to and changing the activity of proteins.

In addition to multichannel and NIR fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor and white light imaging of blots, gels, tissues, and more. Download a free copy of the Sapphire Applications Booklet and learn about how you can add more applications to your arsenal here.

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A New Mechanism by Which Bacteriophage T5 Inhibits Growth of E. coli

Categories
Protein Assays Quantification

How can we better control pathogenic bacteria? Insights may come from studying bacteriophages, viruses that infect bacteria. There are a wide variety of bacteriophages, each of which is specialized to infect and replicate within a specific target bacteria. Learning how a bacteriophage takes over bacterial metabolism to direct resources towards generating more bacteriophage can both increase understanding of bacterial metabolism, and potentially provide ideas for new antibiotics or new means of controlling bacterial pathogens.

In recent work, Mahata et al from Tel Aviv University in Israel developed a high-throughput sequencing approach to identify functions for T5 proteins. The bacteria were mutagenized and then screened to identify bacterial mutants that were resistant to growth inhibition by the phage protein T5.015.

To demonstrate the DNA cleavage activity of T5.015, the Azure Sapphire™ Biomolecular Imager was used to detect cleavage products of Cy5.5-labeled oligomers separated by gel electrophoresis (Figure 1). High-throughput sequencing of the mutants characterized the DNA changes responsible for the resistance. The researchers found mutations in the ung gene made the bacteria resistant to the effects of T5.015.

Cy5.5 fluorescence of assay products from Azure Sapphire Biomolecular Imager resolved on denaturing urea polyacrylamide gel are shown.
Figure 1. 015 cleaves Ung-generated AP sites. (A) Schematic diagram of the Ung-generated AP site cleavage assay. (B and C) AP site cleavage assay performed in the presence of the Cy5.5-labeled dsDNA oligomer and the indicated purified proteins. In B, 33.4 nM (1 U) of Ung, 6.6 nM (2 U) of Ape1, 1.26 µM of 015, and 820 nM (2 U) of Ugi were used. In C, 315 nM of 015 and 6.6 nM of Ape1 were used. Cy5.5 fluorescence of assay products resolved on denaturing urea polyacrylamide gel are shown.

Ung, the protein encoded by the ung gene, is involved in uracil excision, removing uracils mistakenly incorporated into DNA. Normally the Ung protein removes the uracil, and the resulting abasic site in the DNA is repaired. However, the researchers found that in T5 infection, after Ung removes an uracil, T5.015 cleaves the DNA at the abasic site. DNA cleavage pauses DNA replication and inhibits bacterial growth. The authors hypothesize that halting DNA replication and cell division makes more resources available to the phage.

Conveniently,­ T5 encodes a dUTPase that reduces UTP levels in the bacteria after infection so newly synthesized phage DNA is much less likely to contain any uracil, and only bacterial DNA is targeted by T5.015. The mechanism identified by Mahata et al represents a previously unknown means of bacterial growth inhibition by a bacteriophage.

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Model of 015-mediated toxicity. T5 bacteriophage translocates its genetic material into the E. coli cell, which expresses 015 within several minutes (1). The 015 gene product forms a complex with the host Ung (2) and thus localizes to newly formed AP sites (3). 015 then attacks the AP site (4) and forms a nick in the chromosomal DNA (5), which leads to DNA replication arrest and ultimately to cell death (6). Licensed under CC BY 4.0

Bacteriophage T5 infects the bacteria Escherichia coli. Some strains of E. coli are found normally in the human gut, but other strains are pathogenic and are responsible for some cases of foodborne illness. T5 is an intriguing bacteriophage because of its large genome which encodes over 160 proteins, only about half of which have known, or proposed functions based on homology. Therefore, studying T5 holds the potential to reveal novel bacterial biological or biochemical mechanisms in addition to providing potential new avenues to controlling pathogens.

In addition to multichannel fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor, near-infrared and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire by clicking here and requesting a demo.

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Investigating S-acylation of SARS-Cov-2 Spike Protein Leads to New Insights into Viral Infectivity

Categories
COVID-19 Fluorescence imaging Imaging Western Blotting

A better understanding of coronavirus biology can enable development of new antivirals to help stop COVID-19 and prevent future pandemics. In recent work, Puthenveetil et al. characterized S-acylation of the Spike (S) protein of SARS-CoV-2. This post-translational modification is known to be important to the viral replication cycle of other viruses across multiple virus families but has not been studied in SARS-CoV-2.

To control for gel loading and S protein expression in the S-acylation experiments, the authors used the Azure Sapphire Biomolecular Imager to detect GAPDH by NIR fluorescence and SARS-CoV-2 S protein by chemiluminescence on Western blots (Figure 1).

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

The authors expressed S protein in cultured cells and carried out metabolic labeling with a fatty acid that was detected using a rhodamine-labeled fluorescent probe. They found substantial S-acylation that was blocked by 2BP, a global inhibitor of S-acylation. The authors also found the S-acylation of the S protein was dependent on the presence of the cysteines in the C-terminal domain.

Sapphire was used to image chemiluminescent and NIR Western blots to control for protein loading and S-protein expression in the S-acylation assay
Figure 1 from Puthenveetil R, Lun CM, Murphy RE, et al. (2021) S-acylation of SARS-CoV-2 Spike protein: mechanistic dissection, in vitro reconstitution and role in viral infectivity. Licensed under CC BY 4.0. The Sapphire was used to image chemiluminescent and NIR Western blots to control for protein loading and S-protein expression in the S-acylation assay (panel B).

Further experiments expressing mutated versions of the S protein identified which cysteines were S-acylated, the effect of mutating these cysteines on particle infectivity, and which members of the human family of enzymes that carry out S-acylation were able to modify the S protein in cells and in an in vitro assay.

What is S-acylation?

S-acylation involves adding long-chain fatty acids to cysteine residues on the cytosolic side of transmembrane proteins. The cytoplasmic tail of the SARS-CoV-2 S protein contains 10 cysteines in 6 potential S-acylation sites. Puthenveetil et al note that all but one of these are conserved with SARS-CoV, and most are conserved with other coronaviruses that infect humans, including MERS. Still, nothing is known about what role, if any, the S-acylation of these cysteines may play in the biology of viruses, such as SARS-COV-2.

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In addition to chemiluminescence and near-infrared fluorescence imaging, the Sapphire provides densitometry, phosphor, multichannel fluorescence, and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.

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