Aptamer-Antibody Chimera Sensors for Sensitive, Rapid, and Reversible Molecular Detection in Complex Samples.

The development of receptors suitable for the continuous detection of analytes in complex, interferent-rich samples remains challenging. Antibodies are highly sensitive but difficult to engineer in order to introduce signaling functionality, while aptamer switches are easy to construct but often yield only a modest target sensitivity. We present here a programmable antibody and DNA aptamer switch (PANDAS), which combines the desirable properties of both receptors by using a nucleic acid tether to link an analyte-specific antibody to an internal strand-displacement (ISD)-based aptamer switch that recognizes the same target through different epitopes. The antibody increases PANDAS analyte binding due to its high affinity, and the effective concentration between the two receptors further enhances two-epitope binding and fluorescent aptamer signaling. We developed a PANDAS sensor for the clotting protein thrombin and show that a tuned design achieves a greater than 300-fold enhanced sensitivity compared to that of using an aptamer alone. This design also exhibits reversible binding, enabling repeated measurements with a temporal resolution of ∼10 min, and retains excellent sensitivity even in interferent-rich samples. With future development, this PANDAS approach could enable the adaptation of existing protein-binding aptamers with modest affinity to sensors that deliver excellent sensitivity and minute-scale resolution in minimally prepared biological specimens.

Small-Gauge Vitrectomy for Macular Surgery Using a Systematic Approach to Wound Closure: 27-Gauge vs 25-Gauge.

Purpose: To evaluate the effectiveness of and to compare vitrectomy performed with 25-gauge or 27-gauge instrumentation for macular surgery by assessing the surgical duration, wound closure, and complication rate using a systematic approach to wound closure. Methods: In this retrospective chart review, 125 25-gauge and 125 27-gauge consecutive small-gauge vitrectomy surgeries for epiretinal membrane, macular hole, vitreomacular adhesion, or a combination were analyzed during and immediately after surgery. Wound closure was performed using a systematic protocol. Results: Baseline characteristics were not statistically different between the 2 groups. The surgical duration was similar with 25-gauge vitrectomy and 27-gauge vitrectomy (P = .07). Although spontaneous wound closure was common in both groups, it was more common in the 27-gauge group (P = .22). Intraoperative and postoperative complications were uncommon in both groups. Conclusions: Findings show that 27-gauge vitrectomy is a safe, effective alternative to the more commonly used 25-gauge vitrectomy for macular surgery. Less manipulation was required to achieve wound closure with 27-gauge vitrectomy using a standardized wound-closure protocol. Smaller 27-gauge vitrectomy did not increase surgical time or complications over 25-gauge vitrectomy for macular surgery.

Modular Aptamer Switches for the Continuous Optical Detection of Small-Molecule Analytes in Complex Media.

Aptamers are a promising class of affinity reagents because signal transduction mechanisms can be built into the reagent, so that they can directly produce a physically measurable output signal upon target binding. However, endowing the signal transduction functionality into an aptamer remains a trial-and-error process that can compromise its affinity or specificity and typically requires knowledge of the ligand binding domain or its structure. In this work, a design architecture that can convert an existing aptamer into a "reversible aptamer switch" whose kinetic and thermodynamic properties can be tuned without a priori knowledge of the ligand binding domain or its structure is described. Finally, by combining these aptamer switches with evanescent-field-based optical detection hardware that minimizes sample autofluorescence, this study demonstrates the first optical biosensor system that can continuously measure multiple biomarkers (dopamine and cortisol) in complex samples (artificial cerebrospinal fluid and undiluted plasma) with second and subsecond-scale time responses at physiologically relevant concentration ranges.

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix.

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time is developed. As a proof of concept, real-time imaging of inter-cellular cyclic adenosine 3',5'-monophosphate (cAMP) signals in Dictyostelium discoideum amoeba cells is performed. A set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals is engineered. By combining multiple switches with different dynamic ranges, measure cAMP concentrations spanning three orders of magnitude in a single experiment can be measured. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements are achieved. Using different aptamer switches, this approach can be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

An antibody-based molecular switch for continuous small-molecule biosensing.

We present a generalizable approach for designing biosensors that can continuously detect small-molecule biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nanomolar to 3.3 millimolar. Last, we integrated this switch with a fiber optic sensor to achieve continuous sensing of cortisol in a buffer and blood with <5-min time resolution. We believe that this modular sensor design can enable continuous biosensor development for many biomarkers.

Opportunities and challenges in the diagnostic utility of dermal interstitial fluid.

The volume of interstitial fluid (ISF) in the human body is three times that of blood. Yet, collecting diagnostically useful ISF is more challenging than collecting blood because the extraction of dermal ISF disrupts the delicate balance of pressure between ISF, blood and lymph, and because the triggered local inflammation further skews the concentrations of many analytes in the extracted fluid. In this Perspective, we overview the most meaningful differences in the make-up of ISF and blood, and discuss why ISF cannot be viewed generally as a diagnostically useful proxy for blood. We also argue that continuous sensing of small-molecule analytes in dermal ISF via rapid assays compatible with nanolitre sample volumes or via miniaturized sensors inserted into the dermis can offer clinically advantageous utility, particularly for the monitoring of therapeutic drugs and of the status of the immune system.

Current Landscape of Uveitis Specialists in the United States.

Purpose This study characterizes the current landscape of uveitis specialists and their practice settings in the United States. Methods An anonymous Internet-based survey with questions pertaining to training history and practice characteristics was distributed via REDCap to the American Uveitis Society and Young Uveitis Specialists listservs. Results Forty-eight uveitis specialists in the United States responded to the survey out of 174 uveitis specialists that identify as practicing in the United States. Twenty-five of 48 respondents (52%) completed an additional fellowship. These additional fellowships ranged from surgical retina (12/25, 48%), cornea (8/25, 32%), and medical retina (4/25, 16%). Two-thirds of uveitis specialists managed their own immunosuppression, while one-third comanaged immunosuppression with rheumatologists. Thirty-three of 48 (69%) maintained a surgical practice. Conclusion This is the first survey of uveitis specialists across the United States to provide understanding into training and practice characteristics. These data will provide insight into career planning, practice building, and assist in resource allocation.

Rational design of aptamer switches with programmable pH response.

Aptamer switches that respond sensitively to pH could enhance control over molecular devices, improving their diagnostic and therapeutic efficacy. Previous designs have inserted pH-sensitive DNA motifs into aptamer sequences. Unfortunately, their performance was limited by the motifs' intrinsic pH-responses and could not be tuned to operate across arbitrary pH ranges. Here, we present a methodology for converting virtually any aptamer into a molecular switch with pH-selective binding properties - in acidic, neutral, or alkaline conditions. Our design inserts two orthogonal motifs that can be manipulated in parallel to tune pH-sensitivity without altering the aptamer sequence itself. From a single ATP aptamer, we engineer pH-controlled target binding under diverse conditions, achieving pH-induced selectivity in affinity of up to 1,000-fold. Importantly, we demonstrate the design of tightly regulated aptamers with strong target affinity over only a narrow pH range. Our approach offers a highly generalizable strategy for integrating pH-responsiveness into molecular devices.

Ocular Adverse Events following Use of Immune Checkpoint Inhibitors for Metastatic Malignancies.

PURPOSE: To report the clinical features, severity, and management of ocular immune-related adverse events (irAEs) in the setting of immune checkpoint inhibitor therapy for metastatic malignancies. METHODS: Retrospective chart review at three tertiary ophthalmology clinics. Electronic medical records were reviewed between 2000 and 2017 for patients with new ocular symptoms while undergoing checkpoint inhibition therapy. RESULTS: Eleven patients were identified. Ocular irAEs ranged from keratoconjunctivitis sicca to Vogt-Koyanagi-Harada-like findings. Average timing of irAEs from starting checkpoint inhibitor therapy was 15.7 weeks. Ocular inflammation was successfully controlled with corticosteroids in most cases, however three patients discontinue treatment as a result of ocular inflammation with decreased visual acuity, two discontinued due to progression of metastatic disease, and one discontinued due to severe systemic irAEs. CONCLUSION: We found a wide spectrum of ocular irAEs associated with immune checkpoint inhibitors. In most cases, ocular AEs did not limit ongoing cancer treatment.

Independent control of the thermodynamic and kinetic properties of aptamer switches.

Molecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. Aptamers are useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be engineered into many nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution can only be achieved at the cost of lower sensitivity or high background. Here, we describe a design strategy that decouples and enables independent control over the thermodynamics and kinetics of aptamer switches. Starting from a single aptamer, we create an array of aptamer switches with effective dissociation constants ranging from 10 µM to 40 mM and binding kinetics ranging from 170 ms to 3 s. Our strategy is broadly applicable to other aptamers, enabling the development of switches suitable for a diverse range of biotechnology applications.

Optical coherence tomography angiography characteristics in diabetic patients without clinical diabetic retinopathy.

PURPOSE: The purpose of this study is to measure optical coherence tomography angiography (OCTA)-defined retinal vascular and choriocapillaris parameters in diabetic patients without clinically evident diabetic retinopathy. METHODS: Retrospective case series of eyes in patients with a documented history of either type 1 or type 2 diabetes mellitus without any clinical signs of diabetic retinopathy seen at the Vanderbilt Eye Institute. All eyes underwent dilated funduscopic examination along with OCTA. OCTA analytics were performed to calculate vessel density, flow area, and foveal avascular zone size. RESULTS: Thirty-seven eyes of 20 patients were included in the analysis. The mean glycated hemoglobin A1c of the 20 patients was 7.2%. All patients underwent dilated funduscopic examination by a fellowship-trained retina specialist. No patients had any overt retinopathy on clinical examination. Fifteen eyes had vascular abnormalities (microaneurysms) present on OCTA. Patients with microaneurysms and patients without microaneurysms were compared. Vessel density of the 6 × 6 mm macular cube images was significantly increased in patients with microaneurysms compared with patients without microaneurysms. There were no other significant differences in OCTA characteristics. CONCLUSION: OCTA can detect microvascular changes not otherwise noted on dilated clinical examination. These pre-clinical findings may facilitate earlier intervention for improved glycemic control and prevention of the onset of clinical retinopathy.

FIVE-YEAR FUNCTIONAL OUTCOMES AFTER EPIRETINAL MEMBRANE SURGERY: A Prospective, Controlled Study.

PURPOSE: To evaluate 5-year functional outcomes involving the inner retina after epiretinal membrane (ERM) surgery. METHODS: The study eye (SE) and fellow eye (FE) of 20 patients undergoing ERM surgery were examined preoperatively and at 3, 12, 24, 36, 48, and 60 months postoperatively. Retinal nerve fiber layer and ganglion cell-inner plexiform layer (GC-IPL) thicknesses were analyzed using spectral domain optical coherence tomography. Humphrey visual field mean deviation, pattern SD, and qualitative changes were assessed and compared over time. RESULTS: Mean GC-IPL thickness in SEs was less than that of FEs at all time points with progressive thinning in SEs after ERM surgery. There was significant thinning of the superotemporal GC-IPL in SEs as compared to FEs at 3 months and 60 months (P < 0.05). Humphrey visual field mean deviation was greater in SEs as compared to FEs but statistically significant only at 0, 12, and 24 months (P < 0.05). Pattern SD increased from baseline in SEs but remained near baseline in FEs. CONCLUSION: Surgical eyes after ERM surgery demonstrated progressive thinning of the GC-IPL and transient worsening trends in Humphrey visual field mean deviation and pattern SD as compared to controls after ERM surgery.

Autoimmune retinopathy.

PURPOSE OF REVIEW: Autoimmune retinopathy (AIR) is an immune-mediated disorder characterized by progressive visual loss, abnormal electroretinographic and visual field findings in the presence of circulating anti-retinal antibodies. This review highlights advances made toward understanding the pathophysiology, clinical manifestations, and trends in the management of AIR. RECENT FINDINGS: The pathophysiology of AIR is likely antibody-mediated. AIR serum autoantibodies are variable in their size and retinal tissue they target and can also be present in healthy controls and multiple autoimmune diseases. Rarely, AIR may be associated with dysregulated self-tolerance mechanisms in the thymus. Despite progress in research, our understanding of AIR remains incomplete. Lack of standardized methods for anti-retinal antibody testing continues to challenge the interpretation of seropositivity. Conventional immunosuppressives have been further studied, and promising immunomodulatory therapies, such as targeted B-cell therapy, have been introduced. Newer imaging modalities such as fundus autofluorescence and spectral domain optical coherence tomography may be helpful in diagnosis, monitoring progression of disease and response to treatment. SUMMARY: AIR is a rare but vision-threatening disease whose pathogenesis is poorly defined. Lack of standardized clinical or laboratory criteria further complicates the diagnosis and management. Despite recent progress, further basic science research into the autoimmune process is needed. Prospective controlled clinical trials with immunomodulatory therapy can help define future treatment paradigms.

The tellurium redox immunomodulating compound AS101 inhibits IL-1ß-activated inflammation in the human retinal pigment epithelium.

PURPOSE: AS101 is a non-toxic organotellurium-IV compound with demonstrated immunomodulating activity in vitro and in vivo. Inflammatory responses are attributed to the pathophysiology of numerous ocular diseases. In this study, we wished to elucidate whether AS101 could mitigate pro-inflammatory activity in human retinal pigment epithelial (RPE) cells, which are heavily involved in ocular immune responses, induced by pro-inflammatory IL-ß activity. METHODS: Primary and transformed RPE cells treated with varying concentrations of AS101 were used in this study. Real-time PCR and ELISA assays were used to detect cytokine/chemokine mRNA expression and protein production. Western blot was used to detect changes in the NFκB pathway. Cell viability and proliferation were detected using a Vi-Cell XR cell counter. To measure the cytoprotective capacity of AS101, cell numbers were compared between cells treated with IL-1ß or lipopolysaccharide (LPS) and cells treated with IL-1ß or LPS in the presence of AS101. RESULTS: AS101 inhibited IL-1ß-induced mRNA expression and protein production of IL-6 and IL-8 in RPE cells. The viability of RPE cells treated with IL-1ß and LPS was unaffected. AS101 slightly inhibited RPE cell growth in the presence of higher levels of IL-1ß. Also, AS101 downregulated the IL-1ß activity by inhibiting the phosphorylation of p65, an NFκB subunit. CONCLUSIONS: The results demonstrate that AS101 reduces IL-1ß-induced inflammatory responses in the RPE. In previous studies, AS101 exhibited therapeutic effects in various disease models and was a safe profile in clinical trials. These results suggest that AS101 may have potent anti-inflammatory potential in the eye and confer the downregulation of RPE inflammatory responses in a pathological environment.

Association of complement factor H tyrosine 402 histidine genotype with posterior involvement in sarcoid-related uveitis.

PURPOSE: To determine whether the complement factor H (CFH) tyrosine 402 histidine (Y402H) variant, recently shown to be associated with age-related macular degeneration (AMD) and multifocal choroiditis, is associated with specific ocular sarcoidosis clinical phenotypes in black and white persons. DESIGN: Case-control study. METHODS: The CFH Y402H polymorphism (rs1061170) was genotyped in 41 subjects with ocular sarcoidosis and 393 control subjects. Allele frequencies in the ocular sarcoidosis cases were compared with controls using chi-square score tests. Genotypic model-based (dominant, recessive, and additive) associations of the rs1061170 allele were tested using multivariate logistic regression. Bayesian information criteria were used to formalize model selection. Genotypes were correlated with disease characteristics and severity of ocular inflammation. RESULTS: The C allele (rs1061170) was found in 35% of controls, but occurred with a significantly higher frequency (48.7%) in ocular sarcoidosis cases (odds ratio, 1.72; 95% confidence interval, 1.09 to 2.78; P = .018). Logistic regression demonstrated an association between rs1061170 and ocular sarcoidosis in 2 of 3 genetic models (additive, P = .0078; recessive, P = .0018). Posterior uveitis and panuveitis were overrepresented significantly in cases with the homozygous variant genotype (CC, 91%; P = .047). The population-attributable risk related to this CFH risk variant was 20%. CONCLUSIONS: The Y402H polymorphism of CFH seems to be associated with ocular sarcoidosis in black and white persons. Carriage of the CFH Y402H polymorphism in both alleles is associated with an increased risk for posterior uveitis and panuveitis presentation. The prognostic importance of this genotype will require prolonged follow-up studies.

Evidence of a Multicopper Oxidase in Mn Oxidation by Gaeumannomyces graminis var. tritici.

ABSTRACT Manganese (Mn) oxidation by the plant-pathogenic fungus Gaeumannomyces graminis var. tritici has been correlated with virulence in take-all disease. The mechanism of Mn oxidation has not, however, been investigated adequately. Research on bacteria and other fungi indicates that Mn oxidation is most often the result of the activity of multicopper oxidases. To determine if G. graminis var. tritici oxidizes Mn by similar means, the Mn oxidizing factor (MOF) produced by G. graminis var. tritici was characterized by cultural, spectrophotometric, and cellulose acetate electrophoresis methods. Based on our results, the MOF is an extracellular enzyme with an estimated molecular weight of 50 to 100 kDa. Electrophoresis and spectrophotometry indicate that the MOF is a multicopper oxidase with laccase activity.

Fungal manganese oxidation in a reduced soil.

Manganese chemistry in soils is a function of complex, competing biotic and abiotic reactions. The role of soil-borne fungi in mediating these reactions is poorly understood. The objective of this article is to document direct observation of fungal Mn oxidation in soil under near in situ conditions, and to isolate, describe and confirm the role of fungi in the observed Mn oxidation, and present a model to explain our observations. We incubated soil under different moisture contents in sample cells designed to allow us to use synchrotron microspectroscopic techniques to analyse areas as small as 38x40 microm2. Mn was redistributed and accumulated in distinct small circular shapes or in dendritic patterns near the air-soil interface when water-saturated soil was incubated for >or=7 days. Mn oxidation did not occur at 3 or 52 degrees C indicating that oxidation was caused by microbial activity. Mn-oxidizing fungi were isolated from the sample cells and cultured on agar. Reinoculation of sterile soil with the Mn-oxidizing isolates resulted in the formation of Mn oxides around fungal hyphae. A model to describe the distinct zonal distribution of Mn oxides in the sample cells is presented. We believe that our data are the first direct observation of Mn oxidation by soil-inhabiting fungi under in situ conditions. Mn-oxidizing fungi may play an underappreciated role in the cycling of Mn in soils.