Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. SERCA is inhibited or stimulated by several membrane micropeptides including another-regulin, dwarf open reading frame, endoregulin, phospholamban (PLB), and sarcolipin. We previously showed that these micropeptides assemble into homo-oligomeric complexes with varying affinity. Here, we tested whether different micropeptides can interact with each other, hypothesizing that coassembly into hetero-oligomers may affect micropeptide bioavailability to regulate SERCA. We quantified the relative binding affinity of each combination of candidates using automated fluorescence resonance energy transfer microscopy. All pairs were capable of interacting with good affinity, similar to the affinity of micropeptide self-binding (homo-oligomerization). Testing each pair at a 1:5 ratio and a reciprocal 5:1 ratio, we noted that the affinity of hetero-oligomerization of some micropeptides depended on whether they were the minority or majority species. In particular, sarcolipin was able to join oligomers when it was the minority species but did not readily accommodate other micropeptides in the reciprocal experiment when it was expressed in fivefold excess. The opposite was observed for endoregulin. PLB was a universal partner for all other micropeptides tested, forming avid hetero-oligomers whether it was the minority or majority species. Increasing expression of SERCA decreased PLB-dwarf open reading frame hetero-oligomerization, suggesting that SERCA-micropeptide interactions compete with micropeptide-micropeptide interactions. Thus, micropeptides populate a regulatory network of diverse protein assemblies. The data suggest that the complexity of this interactome increases exponentially with the number of micropeptides that are coexpressed in a particular tissue.

T-cell activation and bacterial infection in skin wounds of recessive dystrophic epidermolysis bullosa patients.

Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell-mediated immunity in RDEB wounds. Using a non-invasive approach for sampling of wound-associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen-specific activation of RDEB wound-derived T cells were investigated by fluorescence-activated cell sorting-based immune-phenotyping and T-cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB-derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound-associated T cells contain populations of CD4+ and CD8+ peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8+ cytotoxic T lymphocytes recognize S. aureus-infected RDEB keratinocytes and respond by producing interleukin-2 (IL-2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB-derived T cells to microbial antigens in vitro does not trigger PD-1-mediated T-cell exhaustion but induces differentiation of the CD4high population into CD4high CD25+ FoxP3+ regulatory T cells. Our data demonstrated that adaptive T cell-mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD-1/Treg-mediated immuno-suppression in RDEB.

Immunological mechanisms underlying progression of chronic wounds in recessive dystrophic epidermolysis bullosa.

Hereditary epidermolysis bullosa (EB) is a mechanobullous skin fragility disorder characterized by defective epithelial adhesion, leading to mechanical stress-induced skin blistering. Based on the level of tissue separation within the dermal-epidermal junction, EB is categorized into simplex (EBS), junctional (JEB), dystrophic (DEB) and Kindler syndrome. There is no cure for EB, and painful chronic cutaneous wounds are one of the major complications in recessive (RDEB) patients. Although RDEB is considered a cutaneous disease, recent data support the underlying systemic immunological defects. Furthermore, chronic wounds are often colonized with pathogenic microbiota, leading to excessive inflammation and altered wound healing. Consequently, patients with RDEB suffer from a painful sensation of chronic, cutaneous itching/burning and an endless battle with bacterial infections. To improve their quality of life and life expectancy, it is important to prevent cutaneous infections, dampen chronic inflammation and stimulate wound healing. A clear scientific understanding of the immunological events underlying the maintenance of chronic poorly healing wounds in RDEB patients is necessary to improve disease management and better understand other wound healing disorders. In this review, we summarize current knowledge of the role of professional phagocytes, such as neutrophils, macrophages and dendritic cells, the role of T-cell-mediated immunity in lymphoid organs, and the association of microbiota with poor wound healing in RDEB. We conclude that RDEB patients have an underlying immunity defect that seems to affect antibacterial immunity.

Intracellular escape strategies of Staphylococcus aureus in persistent cutaneous infections.

Pathogenic invasion of Staphylococcus aureus is a major concern in patients with chronic skin diseases like atopic dermatitis (AD), epidermolysis bullosa (EB), or chronic diabetic foot and venous leg ulcers, and can result in persistent and life-threatening chronic non-healing wounds. Staphylococcus aureus is generally recognized as extracellular pathogens. However, S. aureus can also invade, hide and persist in skin cells to contribute to wound chronicity. The intracellular life cycle of S. aureus is currently incompletely understood, although published studies indicate that its intracellular escape strategies play an important role in persistent cutaneous infections. This review provides current scientific knowledge about the intracellular life cycle of S. aureus in skin cells, which can be classified into professional and non-professional antigen-presenting cells, and its strategies to escape adaptive defense mechanisms. First, we discuss phenotypic switch of S. aureus, which affects intracellular routing and degradation. This review also evaluates potential intracellular escape mechanism of S. aureus to avoid intracellular degradation and antigen presentation, preventing an immune response. Furthermore, we discuss potential drug targets that can interfere with the intracellular life cycle of S. aureus. Taken together, this review aimed to increase scientific understanding about the intracellular life cycle of S. aureus into skin cells and its strategies to evade the host immune response, information that is crucial to reduce pathogenic invasion and life-threatening persistence of S. aureus in chronic cutaneous infections.

Human recombinant relaxin-2 does not attenuate hypertension or renal injury but exacerbates vascular dysfunction in a female mouse model of SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease that disproportionately affects women of reproductive age and increases their risk for developing hypertension, vascular, and renal disease. Relaxin has potential beneficial therapeutic effects in cardiovascular disease through direct actions on the vasculature. The potential therapeutic benefit of relaxin on SLE-associated cardiovascular and renal risk factors like hypertension has not previously been tested. We hypothesized that relaxin would attenuate hypertension, renal injury, and vascular dysfunction in an established female mouse model of SLE (NZBWF1 mice). Serelaxin (human recombinant relaxin-2, 0.5 mg·kg-1·day-1) or vehicle was administered via osmotic mini-pump for 4 wk in female control (NZW) or SLE mice between 28 and 31 wk of age. Serelaxin treatment increased uterine weights in both groups, suggesting that the Serelaxin was bioactive. Mean arterial pressure, measured by carotid artery catheter, was significantly increased in vehicle-treated SLE mice compared with vehicle-treated controls, but was not changed by Serelaxin treatment. Albumin excretion rate, measured by ELISA, was similar between vehicle- and Serelaxin-treated SLE mice and between vehicle- and Serelaxin-treated control mice. Wire myography was performed using isolated carotid arteries to assess endothelial-independent and -dependent vasodilation, and data confirm that SLE mice have impaired endothelium-independent and -dependent relaxation compared with control mice. Serelaxin treatment did not affect endothelium-independent vasodilation, but exacerbated the endothelium-dependent dysfunction. These data suggest that, contrary to our hypothesis, Serelaxin infusion does not attenuate hypertension, renal injury, or vascular dysfunction in SLE, but worsens underlying vascular endothelial dysfunction in this experimental model of SLE. These data do not support the use of human recombinant relaxin-2 as an antihypertensive in the SLE patient population. NEW & NOTEWORTHY Relaxin is a peptide hormone commonly known for its role in pregnancy and for its use in recent clinical trials for the treatment of heart failure. Evidence suggests that relaxin has immunomodulatory effects; however, the potential therapeutic impact of relaxin in chronic immune mediated disease is unclear. This study tests whether recombinant human relaxin (Serelaxin) attenuates the progression of autoimmunity, and the associated cardiovascular consequences, in an experimental model of systemic lupus erythematosus.

Beacons Contribute Valuable Empirical Information to Theoretical 3-D Aptamer-Peptide Binding.

DNA aptamers were developed against five different peptides from the known binding regions of anti-Cytomegalovirus and anti-Herpes Simplex Virus-2 antibodies and the aptamers were ranked by relative affinity based on an ELISA-like (ELASA) microplate assay. The secondary structures of the top five highest affinity aptamers were studied for stem-loop commonalities and the most probable peptide binding sites. Two of these stem-loop structures were converted into beacons by addition of TYE 665 dye on the 5' end and Iowa Black quencher on the 3' end. When competed against increasing concentrations of each of the five peptides, only three of the possible ten interactions demonstrated "lights on" fluorescence beacon responses. When modeled by generation of PDB files, after passage through PATCHDOCK and YASARA, two of the aptamer beacon-peptide interactions showed no theoretical evidence of separating the G-C stem-loop region, despite clear empirical evidence of separation of the fluorophore and quencher beyond the Förster distance leading to abundant fluorescence. And in the second beacon's case, YASARA modeling suggested that the beacon was always open despite clear empirical evidence that it was not (no fluorescence response) and only opened in the presence of one of the five peptides. These results are interpreted as a demonstration that 3-dimensional docking software such as PATCHDOCK and YASARA, which are based on rigid receptor-ligand shape complementarity may not reflect the "induced-fit" interactions between aptamers and their cognate targets. Therefore, for the most complete and accurate picture of aptamer-peptide binding, several theoretical and empirical (e.g., beacon fluorescence) analysis methods may be needed.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid listeria detection.

A fluorescent DNA aptamer-magnetic bead sandwich assay was developed to detect listeriolysin O (LLO) protein from pathogenic Listeria bacteria using a peroxidase-linked system, Amplex Ultra Red (AUR; derivatized resazurin) substrate, and a custom-designed handheld fluorometer. The assay is highly sensitive with demonstrated limits of detection (LODs) in the range of 4 to 61 L. monocytogenes cells or the equivalent LLO produced by 4 to 61 cells on average in separate titration trials. Total assay processing and analysis time was approximately 30 mins. The assay has demonstrated the ability to detect 6 species of Listeria as desired by the USDA's Food Safety Inspection Service (FSIS). The portable system was designed to be used primarily with surface swab samples from fomites, but it can also be used to assess enrichment cultures. The minimal time to detect a positive enrichment culture in our hands from an initial 10 cell inoculum in 200 ml of broth has been 8 h post-incubation at 37 °C in shaker flask cultures. An optional automated magnetic bead assay processing and wash device capable of simultaneously processing 6 samples with low and consistent fluorescence background for higher volume central laboratories is also described.

Emolabeling increases healthy food choices among grade school children in a structured grocery aisle setting.

Health literacy, the ability to acquire health-related knowledge and make appropriate health-related decisions, is regarded as a key barrier to meaningfully convey health information to children and can impact food choice. Emolabeling is an image-based labeling strategy aimed at addressing this problem by conveying health information using emotional correlates of health using emoticons (happy = healthy; sad = not healthy). To test the utility of such a method to promote healthy food choices among children, 64 children (59% girls, <5% non-White, mean BMI = 52nd percentile) in kindergarten through 5th grade were first given a brief 5-min lesson on how to use the emoticons, then asked to choose any 4 foods in each of 2 aisles structured to mimic a grocery aisle - there were 12 identical foods placed in the same location in each aisle with half being low calorie and half high calorie snacks. Foods were emolabeled in one aisle; no emolabels were used in the other aisle; the order that children were brought in each aisle was counterbalanced. Results showed that adding emolabels increased the number (M ± SD) of healthy foods chosen (3.6 ± 0.7 with vs. 2.3 ± 1.1 without emolabels present [95% CI 1.0, 1.5], R(2) = .67) and reduced the total calories (M ± SD) of foods chosen (193.5 ± 88.5 Cal with vs. 374.3 ± 152.6 Cal without emolabels present [95% CI -212.6, -149.0], R(2) = .70). Hence, adding emolabels was associated with healthier food choices among children, thereby demonstrating one possible strategy to effectively overcome health literacy barriers at these ages.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies.

A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼ 100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.

Preliminary Development of a DNA Aptamer-Magnetic Bead Capture Electrochemiluminescence Sandwich Assay for Brain Natriuretic Peptide.

Fifty-two candidate DNA aptamer sequences were selected for binding to the cardiovascular biomarker B-type or brain natriuretic peptide (BNP). Candidate aptamers were screened to rank their relative affinities against BNP by an aptamer-based ELISA-like aptamer microplate assay (ELASA). The highest affinity aptamers from ELASA screening were also paired in all possible combinations and screened for electrochemiluminescence (ECL) assay potential in capture aptamer-magnetic bead and ruthenium trisbipyridine (Ru(bpy)32+)-reporter aptamer sandwich formats. The top ECL sandwich combinations utilized the same aptamer pair in either capture or reporting roles with nanogram to low picogram per mL levels of detection even in 50% human serum. ECL assay sensitivity and linearity even in 50% human serum suggest that the aptamer-based assay is at least comparable to other reported immunoassays for BNP.

Toward more diverse, inclusive, and equitable neuromodulation.

Racial and ethnic disparities exist for many nervous system disorders that are intervention targets for neuromodulation investigators. Yet, to date, there has been both a lack of racial and ethnic diversity and a lack of emphasis on diversity in neuromodulation research. In this paper, we suggest three potential reasons for the lack of racial and ethnic diversity in neuromodulation research: 1) the lack of diversity in the neuromodulation workforce, 2) incompatibility between the technologies employed and phenotypic traits (e.g., hair texture) commonly present in minoritized populations, and 3) minoritized populations' reluctance to participate in clinical trials. We argue that increasing diversity in the neuromodulation workforce, in conjunction with mutual collaboration between current neuromodulation researchers and underrepresented communities in neuromodulation, can aid in removing barriers to diversity, equity, and inclusion in neuromodulation research. This is important, because greater diversity, equity, and inclusion in neuromodulation research brings with it the development of novel, yet safe and effective, treatment approaches for brain disorders and enhances the rigor and generalizability of discoveries in the field.

Perceived antidepressant efficacy associated with reduced negative and enhanced neutral mnemonic discrimination.

Introduction: While antidepressants are one of the first-line treatments for depression, the mechanisms underlying antidepressant action are unclear. Furthermore, the extent to which antidepressants impact emotional and cognitive dysfunction in depression requires more fine-grained approaches toward measuring these impacts in humans. Depression is associated with emotion and mood dysregulation in addition to cognitive deficits. Depressed individuals experience general memory impairment as well as a negativity bias in episodic memory, where negative events are better remembered than positive or neutral events. One potential mechanism hypothesized to underlie the negativity bias in memory is dysfunctional hippocampal pattern separation, in which depressed individuals tend to show impaired general pattern separation but enhanced negative pattern separation. Mnemonic discrimination tasks have been designed to tax hippocampal pattern separation in humans and provide a powerful approach to develop a mechanistic account for cognitive dysfunction in depression. While antidepressants have been examined primarily in rodent models in the context of hippocampal pattern separation, this has yet to be examined in humans. Methods: Here, we investigated how antidepressant usage and their perceived efficacy was associated with emotional mnemonic discrimination, given our prior work indicating a negativity bias for mnemonic discrimination in individuals with greater depressive symptoms. Results: We found that individuals who reported a greater improvement in their depressive symptoms after taking antidepressants (responders) showed reduced negative and enhanced neutral mnemonic discrimination compared to those with little to no improvement (non-responders). Perceived antidepressant efficacy was the strongest predictor of a reduction in the negativity bias for mnemonic discrimination, even when controlling for current depressive symptoms, antidepressant type, and other relevant factors. Discussion: These results suggest that antidepressants, when effective, can shift memory dynamics toward healthy function.

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.

The sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca 2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB when compared to WT-PLB. In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation stabilizes PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca 2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca 2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

Serum inverts and improves the fluorescence response of an aptamer beacon to various vitamin D analytes.

A dominant aptamer loop structure from a library of nearly 100 candidate aptamer sequences developed against immobilized 25-hydroxyvitamin D(3) (calcidiol) was converted into a 5'-TYE 665 and 3'-Iowa black-labelled aptamer beacon. The aptamer beacon exhibited a mild 'lights on' reaction in buffer as a function of increasing concentrations of several vitamin D analogues and metabolites, with a limit of detection of approximately 200 ng/mL, and was not specific for any particular congener. In 10% or 50% human serum, the same aptamer beacon inverted its fluorescence behaviour to become a more intense 'lights off' reaction with an improved limit of detection in the range 4-16 ng/mL. We hypothesized that this drastic change in fluorescence behaviour was due to the presence of creatinine and urea in serum, which might destabilize the quenched beacon, causing an increase in fluorescence followed by decreasing fluorescence as a function of vitamin D concentrations that may bind and quench increasingly greater fractions of the denatured beacons. However, the results of several control experiments in the presence of physiological or greater concentrations of creatinine and urea, alone or combined in buffer, failed to produce the beacon fluorescence inversion. Other possible mechanistic hypotheses are also discussed.

Regulation of the regulator: Endopeptidase cleavage controls the expression of a micropeptide that regulates SERCA.

Micropeptides regulate cellular calcium handling by modulating the function of the calcium transporter SERCA. In a recent Nature Communications paper [4] authors Schiemann et al. describe regulation of an invertebrate SERCA-active micropeptide, sarcolamban, by an endopeptidase called neprilysin 4 (NEP4). NEP4 activity limits sarcolamban expression by cleavage of luminal residues near the micropeptide's c-terminus. This cleavage event liberates sarcolamban from the membrane, reduces its oligomerization, and prevents it from inhibiting SERCA. The study reveals a novel mechanism for "regulation of the regulator" that may be a general feature of micropeptide/SERCA physiology.

DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

A novel screening method for competitive FRET-aptamers applied to E. coli assay development.

A novel high-throughput screening method is described in which a family of DNA aptamers selected against E. coli outer membrane proteins (OMPs) is subjected to PCR in the presence of fluorophore-dUTP conjugates using Deep Vent® exo- polymerase. The fluorophore-doped aptamers and their complementary strands are then heated to render them single-stranded and screened in filter well microtiter plates for fluorescence resonance energy transfer (FRET) assay potential. Using this system, a superior competitive FRET-aptamer designated EcO 4R was identified and the location of its putative binding pocket was determined by individually testing FRET potential in each of the secondary loop structures. By labeling the binding pocket with Alexa Fluor (AF) 647 and binding the aptamer to heavily Black Hole Quencher-3 (BHQ-3)-labeled E. coli bacteria, detection of as few as 30 live unlabeled E. coli per ml was achieved in a competitive displacement FRET assay format. The far red fluorescence emission enables detection in largely blue-green autofluorescent matrices. In addition, the competitive transfer of AF 647-EcO-4R aptamer to unlabeled E. coli cells after a 15 min equilibration period was verified by fluorescence microscopy. The present study also demonstrated that high aptamer affinity is not well correlated with competitive FRET potential.

Aberrant recruitment of leukocytes defines poor wound healing in patients with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Poorly healing wounds are one of the major complications in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB). At present, there are no effective means to analyze changes in cellular and molecular networks occurring during RDEB wound progression to predict wound outcome and design betted wound management approaches. OBJECTIVES: To better define mechanisms influencing RDEB wound progression by evaluating changes in molecular and cellular networks. METHODS: We developed a non-invasive approach for sampling and analysis of wound-associated constituents using wound-covering bandages. Cellular and molecular components from seventy-six samples collected from early, established and chronic RDEB wounds were evaluated by FACS-based immuno-phenotyping and ELISA. RESULTS: Our cross-sectional analysis determined that progression of RDEB wounds to chronic state is associated with the accumulation (up to 90 %) of CD16+CD66b+ mature neutrophils, loss of CD11b+CD68+ macrophages, and a significant increase (up to 50 %) in a number of CD11c+CD80+CD86+ activated professional antigen presenting cells (APC). It was also marked by changes in activated T cells populations including a reduction of CD45RO+ peripheral memory T cells from 80 % to 30 % and an increase (up to 70 %) in CD45RA+ effector T cells. Significantly higher levels of MMP9, VEGF-A and cathepsin G were also associated with advancing of wounds to poorly healing state. CONCLUSIONS: Our data demonstrated that wound-covering bandages are useful for a non-invasive sampling and analysis of wound-associated constituents and that transition to poorly healing wounds in RDEB patients as associated with distinct changes in leukocytic infiltrates, matrix-remodeling enzymes and pro-angiogenic factors at wound sites.

DNA aptamers developed against a soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobic groups.

Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para-aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867-876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p-aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library.