Evidence of Pericyte Damage in a Cognitively Normal Cohort: Association With CSF and PET Biomarkers of Alzheimer Disease.

BACKGROUND: Blood-brain barrier (BBB) dysfunction is emerging as an important pathophysiologic factor in Alzheimer disease (AD). Cerebrospinal fluid (CSF) platelet-derived growth factor receptor-ß (PDGFRß) is a biomarker of BBB pericyte injury and has been implicated in cognitive impairment and AD. METHODS: We aimed to study CSF PDGFRß protein levels, along with CSF biomarkers of brain amyloidosis and tau pathology in a well-characterized population of cognitively unimpaired individuals and correlated CSF findings with amyloid-PET positivity. We performed an institutional review board (IRB)-approved cross-sectional analysis of a prospectively enrolled cohort of 36 cognitively normal volunteers with available CSF, Pittsburgh compound B PET/CT, Mini-Mental State Exam score, Global Deterioration Scale, and known apolipoprotein E ( APOE ) ε4 status. RESULTS: Thirty-six subjects were included. Mean age was 63.3 years; 31 of 36 were female, 6 of 36 were amyloid-PET-positive and 12 of 36 were APOE ε4 carriers. We found a moderate positive correlation between CSF PDGFRß and both total Tau (r=0.45, P =0.006) and phosphorylated Tau 181 (r=0.51, P =0.002). CSF PDGFRß levels were not associated with either the CSF Aß42 or the amyloid-PET. CONCLUSIONS: We demonstrated a moderate positive correlation between PDGFRß and both total Tau and phosphorylated Tau 181 in cognitively normal individuals. Our data support the hypothesis that BBB dysfunction represents an important early pathophysiologic step in AD, warranting larger prospective studies. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00094939.

Mammalian cells internalize bacteriophages and use them as a resource to enhance cellular growth and survival.

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.

Development of High Throughput Microscope Mode Secondary Ion Mass Spectrometry Imaging.

This paper describes the development and initial results from a secondary ion mass spectrometer coupled with microscope mode detection. Stigmatic ion microscope imaging enables us to decouple the primary ion (PI) beam focus from spatial resolution and is a promising route to attaining higher throughput for mass spectrometry imaging (MSI). Using a commercial C60+ PI beam source, we can defocus the PI beam to give uniform intensity across a 2.5 mm2 area. By coupling the beam with a position-sensitive spatial detector, we can achieve mass spectral imaging of positive and negative secondary ions (SIs), which we demonstrate using samples comprising metals and dyes. Our approach involves simultaneous desorption of ions across a large field of view, enabling mass spectral images to be recorded over an area of 2.5 mm2 in a matter of seconds. Our instrument can distinguish spatial features with a resolution of better than 20 µm, and has a mass resolution of >500 at 500 u. There is considerable scope to improve this, and through simulations we estimate the future performance of the instrument.

Patient Mealtime Experience: Capturing Patient Perceptions Using a Novel Patient Mealtime Experience Tool.

INTRODUCTION: The aim of this study is to describe the mealtime experience using the qualitative components of the Austin Health Patient Mealtime Experience Tool (AHPMET) to complement the quantitative findings of this tool. METHODS: A multiphase, cross-sectional study was undertaken across all sites of Austin Health (Victoria, Australia) between March 2020 and November 2021. Patient mealtime experience was measured using the AHPMET. Descriptive statistics and a deductive thematic analysis approach described the patients' mealtime experiences. RESULTS: Questionnaire data were collected from 149 participants. Patients were most satisfied with staff interactions, and least satisfied with dimensions of food quality, specifically, flavour, presentation, and menu variety. Clinical symptoms, nutrition impact symptoms and the patient's position were barriers to consumption. DISCUSSION: Food quality was perceived as the poorest aspect of patient satisfaction with the hospital foodservice, particularly flavour, presentation, and menu variety. Future foodservice quality improvements must prioritise improving food quality to have the greatest impact on patient satisfaction. While clinical and organisational systems have a role in improving mealtime experience and oral intake, communicating patient perceptions of the mealtime experience is critical for responding to current perceptions of hospital food quality. CONCLUSION: Mealtime experience in the hospital has a significant impact on oral intake and patients' wider perception of hospital services. Questionnaires have been used to capture patient satisfaction with foodservice in the hospital; however, no comprehensive questionnaires including qualitative questions that capture the broader mealtime experience have been validated across different hospital settings. The tool developed through this study can be implemented in any acute and subacute health service to provide feedback and improve the mealtime experience of patients. This has the capacity to improve mealtime intake, mitigate malnutrition, and improve quality of life and patient outcomes.

Dissemination of public health research to prevent non-communicable diseases: a scoping review.

BACKGROUND: Dissemination is a critical element of the knowledge translation pathway, and a necessary step to ensure research evidence is adopted and implemented by key end users in order to improve health outcomes. However, evidence-based guidance to inform dissemination activities in research is limited. This scoping review aimed to identify and describe the scientific literature examining strategies to disseminate public health evidence related to the prevention of non-communicable diseases. METHODS: Medline, PsycInfo and EBSCO Search Ultimate were searched in May 2021 for studies published between January 2000 and the search date that reported on the dissemination of evidence to end users of public health evidence, within the context of the prevention of non-communicable diseases. Studies were synthesised according to the four components of Brownson and colleagues' Model for Dissemination of Research (source, message, channel and audience), as well as by study design. RESULTS: Of the 107 included studies, only 14% (n = 15) directly tested dissemination strategies using experimental designs. The remainder primarily reported on dissemination preferences of different populations, or outcomes such as awareness, knowledge and intentions to adopt following evidence dissemination. Evidence related to diet, physical activity and/or obesity prevention was the most disseminated topic. Researchers were the source of disseminated evidence in over half the studies, and study findings/knowledge summaries were more frequently disseminated as the message compared to guidelines or an evidence-based program/intervention. A broad range of dissemination channels were utilised, although peer-reviewed publications/conferences and presentations/workshops predominated. Practitioners were the most commonly reported target audience. CONCLUSIONS: There is a significant gap in the peer reviewed literature, with few experimental studies published that analyse and evaluate the effect of different sources, messages and target audiences on the determinants of uptake of public health evidence for prevention. Such studies are important as they can help inform and improve the effectiveness of current and future dissemination practices in public health contexts.

Ion Microscope Imaging Mass Spectrometry Using a Timepix3-Based Optical Camera.

Ion microscopy allows for high-throughput mass spectrometry imaging. In order to resolve congested mass spectra, a high degree of timing precision is required from the microscope detector. In this paper we present an ion microscope mass spectrometer that uses a Timepix3 hybrid pixel readout with an optimal 1.56 ns resolution. A novel triggering technique is also employed to remove the need for an external time-to-digital converter (TDC) and allow the experiment to be performed using a low-cost and commercially available readout system. Results obtained from samples of rhodamine B demonstrate the application of multimass imaging sensors for microscope mass spectrometry imaging with high mass resolution.

Statistical Parametric Mapping in Amyloid Positron Emission Tomography.

Alzheimer's disease (AD), the most common cause of dementia, has limited treatment options. Emerging disease modifying therapies are targeted at clearing amyloid-ß (Aß) aggregates and slowing the rate of amyloid deposition. However, amyloid burden is not routinely evaluated quantitatively for purposes of disease progression and treatment response assessment. Statistical Parametric Mapping (SPM) is a technique comparing single-subject Positron Emission Tomography (PET) to a healthy cohort that may improve quantification of amyloid burden and diagnostic performance. While primarily used in 2-[18F]-fluoro-2-deoxy-D-glucose (FDG)-PET, SPM's utility in amyloid PET for AD diagnosis is less established and uncertainty remains regarding optimal normal database construction. Using commercially available SPM software, we created a database of 34 non-APOE ε4 carriers with normal cognitive testing (MMSE > 25) and negative cerebrospinal fluid (CSF) AD biomarkers. We compared this database to 115 cognitively normal subjects with variable AD risk factors. We hypothesized that SPM based on our database would identify more positive scans in the test cohort than the qualitatively rated [11C]-PiB PET (QR-PiB), that SPM-based interpretation would correlate better with CSF Aß42 levels than QR-PiB, and that regional z-scores of specific brain regions known to be involved early in AD would be predictive of CSF Aß42 levels. Fisher's exact test and the kappa coefficient assessed the agreement between SPM, QR-PiB PET, and CSF biomarkers. Logistic regression determined if the regional z-scores predicted CSF Aß42 levels. An optimal z-score cutoff was calculated using Youden's index. We found SPM identified more positive scans than QR-PiB PET (19.1 vs. 9.6%) and that SPM correlated more closely with CSF Aß42 levels than QR-PiB PET (kappa 0.13 vs. 0.06) indicating that SPM may have higher sensitivity than standard QR-PiB PET images. Regional analysis demonstrated the z-scores of the precuneus, anterior cingulate and posterior cingulate were predictive of CSF Aß42 levels [OR (95% CI) 2.4 (1.1, 5.1) p = 0.024; 1.8 (1.1, 2.8) p = 0.020; 1.6 (1.1, 2.5) p = 0.026]. This study demonstrates the utility of using SPM with a "true normal" database and suggests that SPM enhances diagnostic performance in AD in the clinical setting through its quantitative approach, which will be increasingly important with future disease-modifying therapies.

A physiotherapy led Soft Tissue Injury Clinic is an efficient and cost effective model of care. A paediatric specific interrupted time series study.

OBJECTIVES: To evaluate the effect of introducing a physiotherapist-led paediatric Soft Tissue Injury Clinic model as an alternative to a medically led Fracture Clinic model for conservative hospital management of soft-tissue injuries on: patient wait times; healthcare resource use; and cost-effectiveness. DESIGN: Interrupted time-series analysis (including consecutive eligible-cases). SETTING: Children's hospital, Australia. PARTICIPANTS: The study included 245 cases (117 Soft Tissue Injury Clinic model sample, 128 Fracture Clinic model sample) of patients (<18 years) who presented to a specialist children's hospital emergency department and diagnosed with a soft tissue injury requiring non-surgical outpatient management. INTERVENTIONS: Patients were referred from the emergency department to either an orthopaedic-led fracture clinic (Fracture Clinic model) or physiotherapist-led clinic (Soft Tissue Injury Clinic model) for follow-up and further management as clinically indicated. MAIN OUTCOME MEASURES: Time from emergency department discharge to commencement of definitive outpatient management (primary); healthcare resource use and costs from hospital funder perspective (secondary) and cost-per-day less waiting (cost-effectiveness). RESULTS: The Soft Tissue Injury Clinic was associated with (mean per-person difference (95%CI), P-value) fewer wait days (-8 (-11, -5) days, P<0.001), fewer orthopaedic costs P<0.001, >99% probability of fewer days waiting, 81% probability of less total cost and 81% probability of dominance (cheaper and fewer days to access definitive care). There were no adverse events in either model. CONCLUSIONS: The physiotherapist-led Soft Tissue Injury Clinic represented a safe and efficient alternative referral pathway for patients presenting to the emergency department with soft tissue injuries requiring conservative management.

Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak.

An outbreak of over 1,000 COVID-19 cases in Provincetown, Massachusetts (MA), in July 2021-the first large outbreak mostly in vaccinated individuals in the US-prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined viral genomic and epidemiological data from 467 individuals, including 40% of outbreak-associated cases. The Delta variant accounted for 99% of cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US overall, likely due to high vaccination rates and a robust public health response.

Diagnostic accuracy of shuttle CT angiography (CTA) and helical CTA in the diagnosis of vasospasm.

PURPOSE: To evaluate the diagnostic accuracy of computed tomography angiography (CTA) acquired with shuttle technique (CTAs) and helical CTA (CTAh) for vasospasm, using digital subtraction angiography (DSA) obtained within 24 h as reference standard. METHODS: Thirty-six patients with suspected vasospasm in the setting of aneurysmal subarachnoid hemorrhage (ASAH, 30/36) or acute inflammatory/infectious conditions (6/36) who underwent CTAs (17/36) or CTAh (19/36) followed by DSA within 24 h were identified retrospectively. Presence of vasospasm in the proximal cerebral arterial segments was assessed qualitatively and semi-quantitatively on CTA and subsequent DSA. Sensitivity, specificity, and receiver operating characteristic (ROC) curves were calculated. Inter-rater variability was assessed using Cohen's kappa. RESULTS: On CTAs, 35% of patients had low and 65% had high vasospasm burden. On CTAh, 37% had low and 63% had high vasospasm burden. ROC analysis demonstrated an AUC of 0.87 for CTAs (95%CI 0.67-1.00, p = 0.015) and 0.88 for CTAh (0.72-1.00, p = 0.028). Cohen's kappa was 0.843 (95%CI 0.548-1.000). Thresholding with Youden's J index, CTAs had a sensitivity of 85.71% (95%CI 48.69 to 99.27) and specificity of 66.67% (35.42 to 87.94). CTAh had sensitivity of 100% (56.55 to 100.00) and specificity of 78.57% (52.41 to 92.43). CONCLUSION: CTAs and CTAh yielded similar sensitivity, specificity, and AUC values on ROC analysis for the detection of vasospasm using DSA as reference standard. Our findings suggest that CTAs is a promising alternative to CTAh especially in patients requiring serial imaging, given its potential advantages of decreased radiation exposure, contrast dose, and cost-effectiveness.

Evidence of transmission from fully vaccinated individuals in a large outbreak of the SARS-CoV-2 Delta variant in Provincetown, Massachusetts.

Multiple summer events, including large indoor gatherings, in Provincetown, Massachusetts (MA), in July 2021 contributed to an outbreak of over one thousand COVID-19 cases among residents and visitors. Most cases were fully vaccinated, many of whom were also symptomatic, prompting a comprehensive public health response, motivating changes to national masking recommendations, and raising questions about infection and transmission among vaccinated individuals. To characterize the outbreak and the viral population underlying it, we combined genomic and epidemiological data from 467 individuals, including 40% of known outbreak-associated cases. The Delta variant accounted for 99% of sequenced outbreak-associated cases. Phylogenetic analysis suggests over 40 sources of Delta in the dataset, with one responsible for a single cluster containing 83% of outbreak-associated genomes. This cluster was likely not the result of extensive spread at a single site, but rather transmission from a common source across multiple settings over a short time. Genomic and epidemiological data combined provide strong support for 25 transmission events from, including many between, fully vaccinated individuals; genomic data alone provides evidence for an additional 64. Together, genomic epidemiology provides a high-resolution picture of the Provincetown outbreak, revealing multiple cases of transmission of Delta from fully vaccinated individuals. However, despite its magnitude, the outbreak was restricted in its onward impact in MA and the US, likely due to high vaccination rates and a robust public health response.

A Conserved TCRß Signature Dominates a Highly Polyclonal T-Cell Expansion During the Acute Phase of a Murine Malaria Infection.

CD4+ αß T-cells are key mediators of the immune response to a first Plasmodium infection, undergoing extensive activation and splenic expansion during the acute phase of an infection. However, the clonality and clonal composition of this expansion has not previously been described. Using a comparative infection model, we sequenced the splenic CD4+ T-cell receptor repertoires generated over the time-course of a Plasmodium chabaudi infection. We show through repeat replicate experiments, single-cell RNA-seq, and analyses of independent RNA-seq data, that following a first infection - within a highly polyclonal expansion - T-effector repertoires are consistently dominated by TRBV3 gene usage. Clustering by sequence similarity, we find the same dominant clonal signature is expanded across replicates in the acute phase of an infection, revealing a conserved pathogen-specific T-cell response that is consistently a hallmark of a first infection, but not expanded upon re-challenge. Determining the host or parasite factors driving this conserved response may uncover novel immune targets for malaria therapeutic purposes.

High-Resolution Ion Microscope Imaging over Wide Mass Ranges Using Electrodynamic Postextraction Differential Acceleration.

A time-dependent postextraction differential acceleration (PEDA) potential was used to temporally focus increasingly heavy ions in a stigmatic imaging mass spectrometer, allowing them to be imaged with high mass and spatial resolutions over a broad mass-to-charge (m/z) range. By applying a linearly rising potential to the ion extraction electrode, sequential m/z ratios were subjected to a changing electric field, allowing their foci to coincide at the detector. Using this approach, at least 75% of the maximum mass resolution was obtained over a 300-600 Da range when the ion microscope was focused around 450 Da, representing more than a 10-fold increase over the conventional single-field PEDA method.

Microscope imaging mass spectrometry with a reflectron.

A time-of-flight microscope imaging mass spectrometer incorporating a reflectron was used to image mass-resolved ions generated from a 270 µm diameter surface. Mass and spatial resolutions of 8100 ± 700 m/Δm and 18 µm ± 6 µm, respectively, were obtained simultaneously by using pulsed extraction differential acceleration ion optical focusing to create a pseudo-source plane for a single-stage gridless reflectron. The obtainable mass resolution was limited only by the response time of the position-sensitive detector and, according to simulations, could potentially reach 30 200 ± 2900 m/Δm. The spatial resolution can be further improved at the expense of the mass resolution to at least 6 µm by increasing the applied extraction field. An event-triggered fast imaging sensor was additionally used to record ion images for each time-of-flight peak resolved during an experimental cycle, demonstrating the high-throughput capability of the instrument.

Stimuli-Responsive Pure Protein Organogel Sensors and Biocatalytic Materials.

Utilizing protein chemistry in organic solvents has important biotechnology applications. Typically, organic solvents negatively impact protein structure and function. Immobilizing proteins via cross-links to a support matrix or to other proteins is a common strategy to preserve the native protein function. Recently, we developed methods to fabricate macroscopic responsive pure protein hydrogels by lightly cross-linking the proteins with glutaraldehyde for chemical sensing and enzymatic catalysis applications. The water in the resulting protein hydrogel can be exchanged for organic solvents. The resulting organogel contains pure organic solvents as their mobile phases. The organogel proteins retain much of their native protein function, i.e., protein-ligand binding and enzymatic activity. A stepwise ethylene glycol (EG) solvent exchange was performed to transform these hydrogels into organogels with a very low vapor pressure mobile phase. These responsive organogels are not limited by solvent/mobile phase evaporation. The solvent exchange to pure EG is accompanied by a volume phase transition (VPT) that decreases the organogel volume compared to that of the hydrogel. Our organogel sensor systems utilize shifts in the particle spacing of an attached two-dimensional photonic crystal (2DPC) to report on the volume changes induced by protein-ligand binding. Our 2DPC bovine serum albumin (BSA) organogels exhibit VPT that swell the organogels in response to the BSA binding of charged ligands like ibuprofen and fatty acids. To our knowledge, this is the first report of a pure protein organogel VPT induced by protein-ligand binding. Catalytic protein organogels were also fabricated that utilize the enzyme organophosphorus hydrolase (OPH) to hydrolyze toxic organophosphate (OP) nerve agents. Our OPH organogels retain significant enzymatic activity. The OPH organogel rate of OP hydrolysis is ∼160 times higher than that of un-cross-linked OPH monomers in a 1:1 ethylene glycol/water mixture.

Mechanisms by Which Organic Solvent Exchange Transforms Responsive Pure Protein Hydrogels into Responsive Organogels.

Responsive pure protein organogel sensors and catalysts are fabricated by replacing the aqueous mobile phase of protein hydrogels with pure ethylene glycol (EG). Exchanging water for EG causes irreversible volume phase transitions (VPT) in bovine serum albumin (BSA) polymers; however, BSA hydrogel and organogel sensors show similar volume responses to protein-ligand binding. This work elucidates the mechanisms involved in this enabling irreversible VPT by examining the protein secondary structure, hydration, and protein polymer morphology. Organogel proteins retain their native activity because their secondary structure and hydration shell are relatively unperturbed by the EG exchange. Conversely, the decreasing solvent quality initiates polymer phase separation to minimize the BSA polymer surface area exposed to EG, thus decreasing distances between BSA polymer strands. These protein polymer morphology changes promote interprotein interactions between BSA polymer strands, which increase the effective polymer cross-link density and prevent organogel swelling as the mobile phase is exchanged back to water.

Adaptive periodicity in the infectivity of malaria gametocytes to mosquitoes.

Daily rhythms in behaviour, physiology and molecular processes are expected to enable organisms to appropriately schedule activities according to consequences of the daily rotation of the Earth. For parasites, this includes capitalizing on periodicity in transmission opportunities and for hosts/vectors, this may select for rhythms in immune defence. We examine rhythms in the density and infectivity of transmission forms (gametocytes) of rodent malaria parasites in the host's blood, parasite development inside mosquito vectors and potential for onwards transmission. Furthermore, we simultaneously test whether mosquitoes exhibit rhythms in susceptibility. We reveal that at night, gametocytes are twice as infective, despite being less numerous in the blood. Enhanced infectiousness at night interacts with mosquito rhythms to increase sporozoite burdens fourfold when mosquitoes feed during their rest phase. Thus, changes in mosquito biting time (owing to bed nets) may render gametocytes less infective, but this is compensated for by the greater mosquito susceptibility.

Arizona's Emergency Medical Services for Children Pediatric Designation System for Emergency Departments.

BACKGROUND: In 2012, a voluntary certification program called Pediatric Prepared Emergency Care (PPEC) was established in Arizona as a system for pediatric emergency preparedness. Emergency medicine and pediatric specialists generated basic, intermediate, and advanced designation criteria. Dedicated medical management by a pediatric emergency specialist is required for advanced centers. Designation follows a site visit, review, and approval by the subcommittee and the Arizona Chapter of the American Academy of Pediatrics. DISCUSSION: Arizona has 5 designated pediatric emergency departments, all of which are in the southeast part of the state. Therefore, a designation system was implemented so that all emergency departments statewide can receive more training, support, and supervision of pediatric care. The goal was to create a self-sustaining network with active participation from member institutions while fostering the pediatric commitment. Since its inception, 39 hospitals and 5 tribal facilities have joined PPEC, equating to 51% of Arizona's emergency facilities. Of the hospitals, 7 are advanced, 6 are intermediate, and 17 are basic centers. In 2015, all of the 9 sites due for recertification were recertified. The multiple tiers allow for mutual accountability, sharing of resources, and improved quality of care for pediatrics in emergency departments statewide. CONCLUSION: PPEC enhances the quality of pediatric emergency preparedness by means of voluntary certification. The primary limitations are sustainability and funding, because an Emergency Medical Services for Children grant has offset the cost until now. The number of member facilities in this designation system is continually growing, and universal recertification shows sustainability.

Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors.

Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5-9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing.