Point-of-Care Molecular Test for the Detection of 14 High-Risk Genotypes of Human Papillomavirus in a Single Tube.

Cervical cancers constitute a large disease burden in developing countries, with the human papillomavirus (HPV) being responsible for most cervical lesions. Many regions in low-resource countries lack adequate access to sensitive point-of-care (POC) screening tools, preventing timely diagnosis and treatment. To reduce screening barriers, we developed a POC HPV molecular test that detects 14 high-risk HPV types in 30 min in a single assay. We introduced innovations to the underlying amplification (recombinase polymerase amplification) and detection methodologies such as improved probe design, reagent lyophilization, and pipette-less processing to increase sensitivity while enabling minimally trained personnel to conduct reproducible testing. Based on 198 clinically derived samples, we demonstrated a sensitivity of 93% and a specificity of 73% compared to an FDA-approved polymerase chain reaction-based clinical method. Our modified pipette-less simplified assay had a sensitivity of 96% and a specificity of 83%. The application of our assay is intended as a near-patient screening tool with further evaluation by a clinician for confirmation.

A Portable, Encapsulated Microbial Whole-Cell Biosensing System for the Detection of Bioavailable Copper (II) in Soil.

A portable, field deployable whole-cell biosensor was developed that can withstand the complex matrices of soil and requires minimal to no sample preparation to monitor bioavailable concentrations of the essential micronutrient copper (II). Conventional measurement of micronutrients is often complex, laboratory-based, and not suitable for monitoring their bioavailable concentration. To address this need, we developed a fluorescence based microbial whole-cell biosensing (MWCB) system encoding for a Cu2+-responsive protein capable of generating a signal upon binding to Cu2+. The sensing-reporting protein was designed by performing circular permutation on the green fluorescent protein (GFP) followed by insertion of a Cu2+ binding motif into the structure of GFP. The design included insertion of several binding motifs and creating plasmids that encoded the corresponding sensing proteins. The signal generated by the sensing-reporting protein is directly proportional to the concentration of Cu2+ in the sample. Evaluation of the resulting biosensing systems carrying these plasmids was performed prior to selection of the optimal fluorescence emitting Cu2+-binding protein. The resulting optimized biosensing system was encapsulated in polyacrylate-alginate beads and embedded in soil for detection of the analyte. Once exposed to the soil, the beads were interrogated to measure the fluorescence signal emitted by the sensing-reporting protein using a portable imaging device. The biosensor was optimized for detection of Cu2+ in terms of selectivity, sensitivity, matrix effects, detection limits, and reproducibility in both liquid and soil matrices. The limit of detection (LoD) of the optimized encapsulated biosensor was calculated as 0.27 mg/L and 1.26 mg/kg of Cu2+ for Cu2+ in solution and soil, respectively. Validation of the portable imaging tools as a potential biosensing device in the field was performed.

Vitamin E: Regulatory Role on Signal Transduction.

Vitamin E modulates signal transduction pathways by several molecular mechanisms. As a hydrophobic molecule located mainly in membranes it contributes together with other lipids to the physical and structural characteristics such as membrane stability, curvature, fluidity, and the organization into microdomains (lipid rafts). By acting as the main lipid-soluble antioxidant, it protects other lipids such as mono- and poly-unsaturated fatty acids (MUFA and PUFA, respectively) against chemical reactions with reactive oxygen and nitrogen species (ROS and RNS, respectively) and prevents membrane destabilization and cellular dysfunction. In cells, vitamin E affects signaling in redox-dependent and redox-independent molecular mechanisms by influencing the activity of enzymes and receptors involved in modulating specific signal transduction and gene expression pathways. By protecting and preventing depletion of MUFA and PUFA it indirectly enables regulatory effects that are mediated by the numerous lipid mediators derived from these lipids. In recent years, some vitamin E metabolites have been observed to affect signal transduction and gene expression and their relevance for the regulatory function of vitamin E is beginning to be elucidated. In particular, the modulation of the CD36/FAT scavenger receptor/fatty acids transporter by vitamin E may influence many cellular signaling pathways relevant for lipid homeostasis, inflammation, survival/apoptosis, angiogenesis, tumorigenesis, neurodegeneration, and senescence. Thus, vitamin E has an important role in modulating signal transduction and gene expression pathways relevant for its uptake, distribution, metabolism, and molecular action that when impaired affect physiological and patho-physiological cellular functions relevant for the prevention of a number of diseases. © 2018 IUBMB Life, 71(4):456-478, 2019.

α-Tocopheryl Phosphate Induces VEGF Expression via CD36/PI3Kγ in THP-1 Monocytes.

The CD36 scavenger receptor binds several ligands and mediates ligand uptake and ligand-dependent signal transduction and gene expression, events that may involve CD36 internalization. Here we show that CD36 internalization in THP-1 monocytes is triggered by α-tocopherol (αT) and more strongly by α-tocopheryl phosphate (αTP) and EPC-K1, a phosphate diester of αTP and L-ascorbic acid. αTP-triggered CD36 internalization is prevented by the specific covalent inhibitor of selective lipid transport by CD36, sulfo-N-succinimidyl oleate (SSO). Moreover, SSO inhibited the CD36-mediated uptake of 14C-labelled αTP suggesting that αTP binding and internalization of CD36 is involved in cellular αTP uptake, whereas the uptake of αT was less affected. Similar to that, inhibition of selective lipid transport of the SR-BI scavenger receptor resulted mainly in reduction of αTP and not αT uptake. In contrast, uptake of αT was mainly inhibited by Dynasore, an inhibitor of clathrin-mediated endocytosis, suggesting that the differential regulatory effects of αTP and αT on signaling may be influenced by their different routes of uptake. Interestingly, αTP and EPC-K1 also reduced the neutral lipid content of THP-1 cells and the phagocytosis of fluorescent Staphylococcus aureus bioparticles. Moreover, induction of the vascular endothelial growth factor (VEGF) promoter activity by αTP occurred via CD36/PI3Kγ/Akt, as it could be inhibited by specific inhibitors of this pathway (SSO, Wortmannin, AS-605240). These results suggest that αTP activates PI3Kγ/Akt signaling leading to VEGF expression in monocytes after binding to and/or transport by CD36, a receptor known to modulate angiogenesis in response to amyloid beta, oxLDL, and thrombospondin. J. Cell. Biochem. 118: 1855-1867, 2017. © 2017 Wiley Periodicals, Inc.

Vitamin E: A Role in Signal Transduction.

Vitamin E modulates the activity of several signal transduction enzymes with consequent alterations of gene expression. At the molecular level, vitamin E may directly bind to these enzymes and compete with their substrates, or it may change their activity by redox regulation. The translocation of several of these enzymes to the plasma membrane is regulated by vitamin E, suggesting the modulation of protein-membrane interactions as a common mechanism for vitamin E action. Enzyme-membrane interactions can be affected by vitamin E by interference with binding to specific membrane lipids or by altering cellular structures such as membrane microdomains (lipid rafts). Moreover, competition by vitamin E for common binding sites within lipid transport proteins may alter the traffic of lipid mediators and thus affect their signaling and enzymatic conversion. In this review, the main effects of vitamin E on enzymes involved in signal transduction are summarized and possible molecular mechanisms leading to enzyme modulation are evaluated.

The rise, the fall and the renaissance of vitamin E.

This review deals with the expectations of vitamin E ability of preventing or curing, as a potent antioxidant, alleged oxidative stress based ailments including cardiovascular disease, cancer, neurodegenerative diseases, cataracts, macular degeneration and more. The results obtained with clinical intervention studies have highly restricted the range of effectiveness of this vitamin. At the same time, new non-antioxidant mechanisms have been proposed. The new functions of vitamin E have been shown to affect cell signal transduction and gene expression, both in vitro and in vivo. Phosphorylation of vitamin E, which takes place in vivo, results in a molecule provided with functions that are in part stronger and in part different from those of the non-phosphorylate compound. The in vivo documented functions of vitamin E preventing the vitamin E deficiency ataxia (AVED), slowing down the progression of non-alcoholic steato-hepatitis (NASH), decreasing inflammation and potentiating the immune response are apparently based on these new molecular mechanisms. It should be stressed however that vitamin E, when present at higher concentrations in the body, should exert antioxidant properties to the extent that its chromanol ring is unprotected or un-esterified.

Induction of VEGF expression by alpha-tocopherol and alpha-tocopheryl phosphate via PI3Kγ/PKB and hTAP1/SEC14L2-mediated lipid exchange.

In several studies, vitamin E has been observed to influence angiogenesis and vasculogenesis. We recently showed that the phosphorylated form of α-tocopherol (αT), α-tocopheryl phosphate (αTP), increases the expression of the vascular endothelial growth factor (VEGF). Thus, αTP may act as an active lipid mediator increasing VEGF expression, angiogenesis, and vasculogenesis. Here, we investigated the molecular signaling mechanisms by which αTP induces VEGF expression using cultured HEK293 cells as model system. αT and more so αTP increased VEGF-promoter activity in a phosphatidylinositol-3-kinase gamma (PI3Kγ)-dependent manner. In contrast, after overexpression of PI3Kγ and/or protein kinase B (PKB), VEGF promoter activity was inhibited by αT and more so by αTP. Inhibition by αT and αTP was dependent on the lipid kinase activity of PI3Kγ, whereas an induction was seen with the protein kinase activity, consistent with a model in which PKB inhibition by αT or αTP occurs only when activated at the plasma membrane and possibly involves a phosphatase such as PHLPP1. PI3Kγ-induced VEGF expression was reduced when the human tocopherol-associated protein 1 (hTAP1/SEC14L2) was overexpressed suggesting formation of an inactive PI3Kγ/hTAP1 heterodimer, that could be reactivated by αT and more so by αTP. We suggest a novel signaling mechanism by which αTP stimulates PI3Kγ activity by stimulating hTAP-mediated phosphatidylinositol exchange and presentation to the enzyme and/or dissociation of an inactive heterodimer. At cellular level, hTAP may act as sensor for intracellular lipid information (location, type, and amount of lipid) and translate it into responses of PI3K-mediated signaling and gene expression.

Differential cellular uptake and metabolism of curcuminoids in monocytes/macrophages: regulatory effects on lipid accumulation.

We have previously shown that curcumin (CUR) may increase lipid accumulation in cultured human acute monocytic leukaemia cell line THP-1 monocytes/macrophages, but that tetrahydrocurcumin (THC), an in vivo metabolite of CUR, has no such effect. In the present study, we hypothesised that the different cellular uptake and/or metabolism of CUR and THC might be a possible explanation for the previously observed differences in their effects on lipid accumulation in THP-1 monocytes/macrophages. Chromatography with tandem MS revealed that CUR was readily taken up by THP-1 monocytes/macrophages and slowly metabolised to hexahydrocurcumin sulphate. By contrast, the uptake of THC was low. In parallel with CUR uptake, increased lipid uptake was observed in THP-1 macrophages but not with the uptake of THC or another CUR metabolite and structurally related compounds. From these results, it is possible to deduce that CUR and THC are taken up and metabolised differently in THP-1 cells, which determine their biological activity. The remarkable differential cellular uptake of CUR, relative to THC and other similar molecules, may imply that the CUR uptake into cells may occur via a transporter.

Enhancing the Bioavailability and Bioactivity of Curcumin for Disease Prevention and Treatment.

Curcumin, a natural polyphenolic component from Curcuma longa roots, is the main bioactive component of turmeric spice and has gained increasing interest due to its proposed anti-cancer, anti-obesity, anti-inflammatory, antioxidant, and lipid-lowering effects, in addition to its thermogenic capacity. While intake from dietary sources such as curry may be sufficient to affect the intestinal microbiome and thus may act indirectly, intact curcumin in the body may be too low (<1 microM) and not sufficient to affect signaling and gene expression, as observed in vitro with cultured cells (10-20 microM). Several strategies can be envisioned to increase curcumin levels in the body, such as decreasing its metabolism or increasing absorption through the formation of nanoparticles. However, since high curcumin levels could also lead to undesired regulatory effects on cellular signaling and gene expression, such studies may need to be carefully monitored. Here, we review the bioavailability of curcumin and to what extent increasing curcumin levels using nanoformulations may increase the bioavailability and bioactivity of curcumin and its metabolites. This enhancement could potentially amplify the disease-preventing effects of curcumin, often by leveraging its robust antioxidant properties.

In vivo regulation of gene transcription by alpha- and gamma-tocopherol in murine T lymphocytes.

Of the 8 different analogues (α-, ß-, γ-, δ-tocopherols and tocotrienols) designated as vitamin E, alpha-tocopherol (α-T) has been mostly studied, together with gamma-tocopherol (γ-T) which is abundant in the US diet. We compared the effect of dietary supplementation with adequate or high doses of α-T or γ-T on the number and type of genes expressed following T cell activation. C57BL/6 mice were fed diets containing adequate (30 ppm) or high (500 ppm) amounts of α-T or γ-T for 4 weeks. Spleen T cells were stimulated ex vivo with plate-bound anti-CD3 and soluble anti-CD28, and gene expression changes were assessed by gene array analysis. The data obtained indicated significant qualitative and quantitative differences between the two analogs in regulating gene expression induced by T cell stimulation. Genes were found uniquely responding to either high α-T (e.g. induced: CD40 ligand, lymphotoxin A) or γ-T (e.g. repressed: poliovirus receptor-related-2). Interestingly, in stimulated T-cells from mice supplemented with high amounts of α-T a bigger number of genes were activated than in mice supplemented with the same amounts of γ-T; under the same conditions γ-T repressed the expression of a number of genes larger than α-T. It is possible that the observed diminution in gene expression in T cells after high γ-T in vivo supplementation modulates inflammation or other T cell mediated functions.

Bioactive dietary components-Anti-obesity effects related to energy metabolism and inflammation.

Obesity is the result of the long-term energy imbalance between the excess calories consumed and the few calories expended. Reducing the intake of energy dense foods (fats, sugars), and strategies such as fasting and caloric restriction can promote body weight loss. Not only energy in terms of calories, but also the specific composition of the diet can affect the way the food is absorbed and how its energy is stored, used or dissipated. Recent research has shown that bioactive components of food, such as polyphenols and vitamins, can influence obesity and its pathologic complications such as insulin resistance, inflammation and metabolic syndrome. Individual micronutrients can influence lipid turnover but for long-term effects on weight stability, dietary patterns containing several micronutrients may be required. At the molecular level, these molecules modulate signaling and the expression of genes that are involved in the regulation of energy intake, lipid metabolism, adipogenesis into white, beige and brown adipose tissue, thermogenesis, lipotoxicity, adipo/cytokine synthesis, and inflammation. Higher concentrations of these molecules can be reached in the intestine, where they can modulate the composition and action of the microbiome. In this review, the molecular mechanisms by which bioactive compounds and vitamins modulate energy metabolism, inflammation and obesity are discussed.

Engineered biosensors for the quorum sensing molecule 3,5-dimethyl-pyrazine-2-ol (DPO) reveal its presence in humans, animals, and bacterial species beyond Vibrio cholerae.

A biosensor was engineered to enable the study of the novel quorum sensing molecule (QSM), 3,5-dimethylpyrazin-2-ol (DPO), employed by Vibrio cholerae to regulate biofilm formation and virulence factor production. Investigations into bacterial quorum sensing (QS), a form of communication based on the production and detection of QSMs to coordinate gene expression in a population dependent manner, offer a unique window to study the molecular underpinnings of microbial behavior and host interactions. Herein, we report the construction of an engineered microbial whole-cell bioluminescent biosensing system that incorporates the recognition of the VqmA regulatory protein of Vibrio cholerae with the bioluminescent reporting signal of luciferase for the selective, sensitive, stable, and reproducible detection of DPO in a variety of samples. Importantly, using our newly developed biosensor our studies demonstrate the detection of DPO in rodent and human samples. Employing our developed biosensor should help enable elucidation of microbial behavior at the molecular level and its impact in health and disease.

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19).

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 â€‹min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P â€‹< â€‹0.05, CI: 78.2-93.8%) and specificity of 93.9% (P â€‹< â€‹0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

The difference in the cellular uptake of tocopherol and tocotrienol is influenced by their affinities to albumin.

Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, ß-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.

Regulatory effects of curcumin on lipid accumulation in monocytes/macrophages.

Recent evidence suggests potential benefits from phytochemicals and micronutrients in protecting against atherosclerosis and inflammation, but the molecular mechanisms of these actions are still unclear. Here, we investigated whether the dietary polyphenol curcumin can modulate the accumulation of lipids in monocytes/macrophages. Curcumin increased the expression of two lipid transport genes, the fatty acids transporter CD36/FAT and the fatty acids binding protein 4 (FABP4/aP2; P < 0.05), leading to increased lipid levels in THP-1 and RAW264.7 monocytes and macrophages (P < 0.05). To investigate the molecular mechanisms involved, we assessed the activity of Forkhead box O3a (FOXO3a), a transcription factor centrally involved in regulating several stress resistance and lipid transport genes. Curcumin increased FOXO3a-mediated gene expression by twofold (P < 0.05), possibly as a result of influencing FOXO3a phosphorylation and nuclear translocation. The curcumin derivative, tetrahydrocurcumin (THC), with similar chemical antioxidant activity as curcumin, did not show any measurable effects. In contrast to the in vitro results, curcumin showed a trend for reduction of lipid levels in peritoneal macrophages in LDL receptor knockout mice fed a high fat diet for 4 months, suggesting additional regulatory mechanisms in vivo. Thus, the up-regulation of FOXO3a activity by curcumin could be a mechanism to protect against oxidant- and lipid-induced damage in the inflammatory cells of the vascular system.

Modulation of CD36-mediated lipid accumulation and senescence by vitamin E analogs in monocytes and macrophages.

The CD36/FAT scavenger receptor/fatty acids transporter regulates cellular lipid accumulation important for inflammation, atherosclerosis, lipotoxicity, and initiation of cellular senescence. Here we compared the regulatory effects of the vitamin E analogs alpha-tocopherol (αT), alpha-tocopheryl phosphate (αTP), and αTP/ßCD (a nanocarrier complex between αTP and ß-cyclodextrin [ßCD]) and investigated their regulatory effects on lipid accumulation, phagocytosis, and senescence in THP-1 monocytes and macrophages. Both, αTP and αTP/ßCD inhibited CD36 surface exposition stronger than αT leading to more pronounced CD36-mediated events such as inhibition of DiI-labeled oxLDL uptake, phagocytosis of fluorescent Staphylococcus aureus bioparticles, and cell proliferation. When compared to ßCD, the complex of αTP/ßCD extracted cholesterol from cellular membranes with higher efficiency and was associated with the delivery of αTP to the cells. Interestingly, both, αTP and more so αTP/ßCD inhibited lysosomal senescence-associated beta-galactosidase (SA-ß-gal) activity and increased lysosomal pH, suggesting CD36-mediated uptake into the endo-lysosomal phagocytic compartment. Accordingly, the observed pH increase was more pronounced with αTP/ßCD in macrophages whereas no significant increase occurred with αT, alpha-tocopheryl acetate (αTA) or ßCD. In contrast to αT and αTA, the αTP molecule is di-anionic at neutral pH, but upon moving into the acidic endo-lysosomal compartment becomes protonated and thus is acting as a base. Moreover, it is expected to be retained in lysosomes since it still carries one negative charge, similar to lysosomotropic drugs. Thus, treatment with αTP or αTP/ßCD and/or inhibition of conversion of αTP to αT as it occurs in aged cells may counteract CD36-mediated overlapping inflammatory, senescent, and atherosclerotic events.

Oxidized LDLs as Signaling Molecules.

Levels of oxidized low-density lipoproteins (oxLDLs) are usually low in vivo but can increase whenever the balance between formation and scavenging of free radicals is impaired. Under normal conditions, uptake and degradation represent the physiological cellular response to oxLDL exposure. The uptake of oxLDLs is mediated by cell surface scavenger receptors that may also act as signaling molecules. Under conditions of atherosclerosis, monocytes/macrophages and vascular smooth muscle cells highly exposed to oxLDLs tend to convert to foam cells due to the intracellular accumulation of lipids. Moreover, the atherogenic process is accelerated by the increased expression of the scavenger receptors CD36, SR-BI, LOX-1, and SRA in response to high levels of oxLDL and oxidized lipids. In some respects, the effects of oxLDLs, involving cell proliferation, inflammation, apoptosis, adhesion, migration, senescence, and gene expression, can be seen as an adaptive response to the rise of free radicals in the vascular system. Unlike highly reactive radicals, circulating oxLDLs may signal to cells at more distant sites and possibly trigger a systemic antioxidant defense, thus elevating the role of oxLDLs to that of signaling molecules with physiological relevance.

Microbial whole-cell biosensors: Current applications, challenges, and future perspectives.

Microbial Whole-Cell Biosensors (MWCBs) have seen rapid development with the arrival of 21st century biological and technological capabilities. They consist of microbial species which produce, or limit the production of, a reporter protein in the presence of a target analyte. The quantifiable signal from the reporter protein can be used to determine the bioavailable levels of the target analyte in a variety of sample types at a significantly lower cost than most widely used and well-established analytical instrumentation. Furthermore, the versatile and robust nature of MWCBs shows great potential for their use in otherwise unavailable settings and environments. While MWCBs have been developed for use in biomedical, environmental, and agricultural monitoring, they still face various challenges before they can transition from the laboratory into industrialized settings like their enzyme-based counterparts. In this comprehensive and critical review, we describe the underlying working principles of MWCBs, highlight developments for their use in a variety of fields, detail challenges and current efforts to address them, and discuss exciting implementations of MWCBs helping redefine what is thought to be possible with this expeditiously evolving technology.

Differential effects of natural and synthetic vitamin E on gene transcription in murine T lymphocytes.

Mice were supplemented with low and high doses of natural and synthetic vitamin E, T cells from the spleen isolated and stimulated with plate-bound anti-CD3 and soluble anti-CD28, and gene expression changes assessed by gene array experiments. The data obtained indicate significant qualitative and quantitative differences between the two vitamin forms in regulating gene expression in response to T-cell stimulation. Marker genes have been found whose expression can be considered significant in establishing the level of, and response to vitamin E for both natural and synthetic vitamin E supplementation; unique markers for synthetic vitamin E supplementation and unique markers for natural vitamin E supplementation have been identified.