SUMOylation of the cardiac sodium channel NaV1.5 modifies inward current and cardiac excitability.

BACKGROUND: Decreased peak sodium current (INa) and increased late sodium current (INa,L), through the cardiac sodium channel NaV1.5 encoded by SCN5A, cause arrhythmias. Many NaV1.5 posttranslational modifications have been reported. A recent report concluded that acute hypoxia increases INa,L by increasing a small ubiquitin-like modifier (SUMOylation) at K442-NaV1.5. OBJECTIVE: The purpose of this study was to determine whether and by what mechanisms SUMOylation alters INa, INa,L, and cardiac electrophysiology. METHODS: SUMOylation of NaV1.5 was detected by immunoprecipitation and immunoblotting. INa was measured by patch clamp with/without SUMO1 overexpression in HEK293 cells expressing wild-type (WT) or K442R-NaV1.5 and in neonatal rat cardiac myocytes (NRCMs). SUMOylation effects were studied in vivo by electrocardiograms and ambulatory telemetry using Scn5a heterozygous knockout (SCN5A+/-) mice and the de-SUMOylating protein SENP2 (AAV9-SENP2), AAV9-SUMO1, or the SUMOylation inhibitor anacardic acid. NaV1.5 trafficking was detected by immunofluorescence. RESULTS: NaV1.5 was SUMOylated in HEK293 cells, NRCMs, and human heart tissue. HyperSUMOylation at NaV1.5-K442 increased INa in NRCMs and in HEK cells overexpressing WT but not K442R-Nav1.5. SUMOylation did not alter other channel properties including INa,L. AAV9-SENP2 or anacardic acid decreased INa, prolonged QRS duration, and produced heart block and arrhythmias in SCN5A+/- mice, whereas AAV9-SUMO1 increased INa and shortened QRS duration. SUMO1 overexpression enhanced membrane localization of NaV1.5. CONCLUSION: SUMOylation of K442-Nav1.5 increases peak INa without changing INa,L, at least in part by altering membrane abundance. Our findings do not support SUMOylation as a mechanism for changes in INa,L. Nav1.5 SUMOylation may modify arrhythmic risk in disease states and represents a potential target for pharmacologic manipulation.

Poro-viscoelastic material parameter identification of brain tissue-mimicking hydrogels.

Understanding and characterizing the mechanical and structural properties of brain tissue is essential for developing and calibrating reliable material models. Based on the Theory of Porous Media, a novel nonlinear poro-viscoelastic computational model was recently proposed to describe the mechanical response of the tissue under different loading conditions. The model contains parameters related to the time-dependent behavior arising from both the viscoelastic relaxation of the solid matrix and its interaction with the fluid phase. This study focuses on the characterization of these parameters through indentation experiments on a tailor-made polyvinyl alcohol-based hydrogel mimicking brain tissue. The material behavior is adjusted to ex vivo porcine brain tissue. An inverse parameter identification scheme using a trust region reflective algorithm is introduced and applied to match experimental data obtained from the indentation with the proposed computational model. By minimizing the error between experimental values and finite element simulation results, the optimal constitutive model parameters of the brain tissue-mimicking hydrogel are extracted. Finally, the model is validated using the derived material parameters in a finite element simulation.

Cognitive bias modification for interpretation (CBM-I) for post-traumatic stress disorder: study protocol of an app-based randomised controlled trial.

INTRODUCTION: Previous studies indicate that computerised trainings implementing cognitive bias modification (CBM) for interpretation bias might be promising treatments for trauma-related cognitive distortions and symptoms. However, results are mixed, which might be related to the implemented task (sentence completion task), setting, or training duration. Within the present study, we aim to evaluate the efficacy and safety of an app-based intervention for interpretation bias using standardised imagery audio scripts, which is designed as a standalone treatment. METHODS AND ANALYSIS: The study is a randomised controlled trial, implementing two parallel arms. 130 patients diagnosed with post-traumatic stress disorder (PTSD) will be allocated to either the intervention group or the waiting-list control group receiving treatment as usual. The intervention consists of 3 weeks of an app-based CBM training for interpretation bias using mental imagery, with three training sessions (20 min) per week. Two months after the last training session, 1 week of booster CBM treatment will be implemented, consisting of three additional training sessions. Outcome assessments will be conducted pretraining, 1 week post-training, 2 months post-training, as well as 1 week after the booster session (approximately 2.5 months after initial training termination). The primary outcome is interpretation bias. Secondary outcomes include PTSD-related cognitive distortions and symptom severity, as well as negative affectivity. Outcome assessment will be conducted by intention-to-treat analysis, as well as per-protocol analysis using linear mixed models. ETHICS AND DISSEMINATION: The study was approved by the Ethics Committee of the State Chamber of Physicians in Baden-Wuerttemberg, Germany (number of approval: F-2022-080). Scientific findings will be published in peer-reviewed journals informing future clinical studies, which focus on the reduction of PTSD-related symptoms using CBM. TRIAL REGISTRATION NUMBER: German Clinical Trials Register (DRKS00030285; https://drks.de/search/de/trial/DRKS00030285).

The role of GPD1L, a sodium channel interacting gene, in the pathogenesis of Brugada Syndrome.

Background: Brugada Syndrome (BrS) is an inherited arrhythmia syndrome in which mutations in the cardiac sodium channel SCN5A (NaV1.5) account for approximately 20% of cases. Mutations in sodium channel-modifying genes may account for additional BrS cases, though BrS may be polygenic given common SNPs associated with BrS have been identified. Recent analysis, however, has suggested that SCN5A should be regarded as the sole monogenic cause of BrS. Objective: We sought to re-assess the genetic underpinnings of BrS in a large mutligenerational family with a putative mutation in GPD1L that affects surface membrane expression of NaV1.5 in vitro. Methods: Fine linkage mapping was performed in the family using the Illumina Global Screening Array. Whole exome sequencing of the proband was performed to identify rare variants and mutations, and Sanger sequencing was used to assay previously-reported risk single nucleotide polymorphsims (SNPs) for BrS. Results: Linkage analysis decreased the size of the previously-reported microsatellite linkage region to approximately 3 Mb. GPD1L-A280V was the only coding non-synonymous variation present at less than 1% allele frequency in the proband within the linkage region. No rare non-synonymous variants were present outside the linkage area in affected individuals in genes associated with BrS. Risk SNPs known to predispose to BrS were overrepresented in affected members of the family. Conclusion: Together, our data suggest GPD1L-A280V remains the most likely cause of BrS in this large multigenerational family. While care should be taken in interpreting variant pathogenicity given the genetic uncertainty of BrS, our data support inclusion of other putative BrS genes in clinical genetic panels.

Tissue-Scale Biomechanical Testing of Brain Tissue for the Calibration of Nonlinear Material Models.

Brain tissue is one of the most complex and softest tissues in the human body. Due to its ultrasoft and biphasic nature, it is difficult to control the deformation state during biomechanical testing and to quantify the highly nonlinear, time-dependent tissue response. In numerous experimental studies that have investigated the mechanical properties of brain tissue over the last decades, stiffness values have varied significantly. One reason for the observed discrepancies is the lack of standardized testing protocols and corresponding data analyses. The tissue properties have been tested on different length and time scales depending on the testing technique, and the corresponding data have been analyzed based on simplifying assumptions. In this review, we highlight the advantage of using nonlinear continuum mechanics based modeling and finite element simulations to carefully design experimental setups and protocols as well as to comprehensively analyze the corresponding experimental data. We review testing techniques and protocols that have been used to calibrate material model parameters and discuss artifacts that might falsify the measured properties. The aim of this work is to provide standardized procedures to reliably quantify the mechanical properties of brain tissue and to more accurately calibrate appropriate constitutive models for computational simulations of brain development, injury and disease. Computational models can not only be used to predictively understand brain tissue behavior, but can also serve as valuable tools to assist diagnosis and treatment of diseases or to plan neurosurgical procedures. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Physical aspects of cortical folding.

The characteristically folded surface of the human brain is critical for brain function and allows for higher cognitive abilities. Recent mostly computational research advances have shown that mechanical instabilities play a crucial role during early brain development and cortical folding. However, it is difficult to investigate such mechanisms in vivo. To experimentally gain deeper insights into the physical mechanisms that underlie the development of brain shape, we use a setup of swelling polymers. We investigate the influence of cortical thickness and the stiffness ratio between cortex and subcortex on the resulting surface pattern by taking the initially smooth fetal brain geometry at week 22 into consideration. The gel specimens possess a two-layered structure accounting for gray and white matter tissue and yield complex surface morphologies that well resemble patterns in the human brain. The results are in good agreement with analytical predictions. Through the variation of cortical thickness and stiffness, it is possible to reproduce cortical malformations such as polymicrogyria and lissencephaly. The results suggest that wrinkling with subsequent transition into folding is the driving instability mechanism during brain development. In addition, the experiments provide valuable insights towards the distinction between wrinkling and creasing instabilities. Taken together, the presented swelling experiments impressively demonstrate the purely physical aspects of brain shape and constitute a valuable tool to advance our understanding of human brain development.

Continuous and Daily Oral Immunotherapy for Peanut Allergy: Results from a 2-Year Open-Label Follow-On Study.

BACKGROUND: The randomized, controlled PALISADE trial demonstrated the benefit of daily oral immunotherapy with Peanut (Arachis Hypogaea) allergen powder-dnfp (PTAH, formerly AR101) in peanut-allergic children and adolescents. OBJECTIVE: ARC004, the open-label follow-on study to PALISADE, used 5 dosing cohorts to explore PTAH treatment beyond 1 year and alternative dosing regimens in peanut-allergic individuals. METHODS: Active arm (PTAH-continuing) PALISADE participants who tolerated 300-mg peanut protein at the exit double-blind placebo-controlled food challenge and placebo arm (PTAH-naive) participants could enter ARC004. PTAH-continuing participants were assigned to receive daily (cohorts 1 and 3A) or non-daily (cohorts 2, 3B, and 3C) dosing regimens; PTAH-naive participants were built up to 300 mg/d PTAH, followed by maintenance dosing. At study completion, participants underwent an exit double-blind placebo-controlled food challenge with doses up to 2000 mg peanut protein. Data were assessed using descriptive statistics. RESULTS: Overall, 358 (87.5%) eligible participants (4-17 years) entered ARC004 (PTAH-continuing, n = 256; PTAH-naive, n = 102). Among PTAH-continuing participants, exposure-adjusted adverse event rates were 12.94 to 17.54/participant-year and 25.95 to 42.49/participant-year in daily and non-daily dosing cohorts, respectively; most participants (83%) experienced mild or moderate adverse events. Daily dosing cohorts appeared to have higher desensitization rates than non-daily dosing cohorts. Of all PTAH-continuing cohorts, cohort 3A had the longest daily dosing duration and the highest desensitization rates. Changes in immune markers with PTAH continuation demonstrated ongoing immunomodulation. Outcomes in PTAH-naive participants mirrored those of the PALISADE active arm. CONCLUSIONS: Continued daily PTAH treatment beyond 1 year showed sustained safety and efficacy. Ongoing immunomodulation was observed during the second year of treatment.

Modulation of the cardiac sodium channel NaV1.5 peak and late currents by NAD+ precursors.

RATIONALE: The cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown. OBJECTIVE: To determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,Lin vitro and cardiac electrophysiology in vivo. METHODS AND RESULTS: The effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 µM: 51 ± 18%, p = .02, 5 mM: 59 ± 22%, p = .03) and RNCMs (500 µM: 60 ± 26%, p = .02, 5 mM: 74 ± 39%, p = .03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: -45 ± 11%, p = .04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6 J mice (0.35% NR: -4.9 ± 2.0%, p = .14; 1.0% NR: -9.5 ± 2.8%, p = .01). CONCLUSIONS: NAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

IDENTIFYING NEW SUDDEN DEATH GENES.

Inherited conditions that lead to cardiac arrhythmias and sudden cardiac death remain an important cause of morbidity and mortality. Identifying the genes responsible for these rare conditions can provide insights into the more common and heritable forms of sudden cardiac death seen in patients with structural heart disease. We and others have used candidate gene approaches and positional cloning in large families to show that mutations in ion channels and ion channel related proteins cause familial arrhythmia syndromes including long QT and Brugada syndromes. The genes responsible for many familial arrhythmia syndromes and the vast majority of the predisposition to common arrhythmias remain unknown. Using whole exome sequencing in families with Brugada syndrome and idiopathic ventricular fibrillation, we now seek to identify mutations in genes previously not thought to play a significant role in the heart.

Rethinking medical oaths using the Physician Charter and ethical virtues.

OBJECTIVE: Medical oaths express ethical values that are essential to the trust within the patient-physician relationship and medicine's commitment to society. However, the contents of oaths vary between medical schools and therefore raise questions about which ethical values should be included in a medical oath. More than a decade has passed since this variability was last analysed in North America, and since that time the Physician Charter on Medical Professionalism has gained considerable attention, raising the possibility that the Charter may be influencing medical oaths and making them more consistent. METHODS: The authors conducted a content analysis of 84 oaths available in 2015 from medical schools in the USA and Canada affiliated with the Association of American Medical Colleges, organising the content into three categories: (i) ethical values, (ii) principles and commitments in the Physician Charter, and (iii) ethical virtues. RESULTS: Only five ethical values were expressed in the majority of oaths (confidentiality, obligation to the profession, beneficence, avoiding discrimination, and honour and integrity), and respect for patient autonomy was uncommon. Only three of the Physician Charter's principles and commitments (primacy of patient welfare, social justice and confidentiality) and one virtue (honour and integrity) were reflected in the majority of oaths. CONCLUSIONS: Medical oaths in North America appear to be highly variable in content. Greater attention to resources like the Physician Charter can help improve the ethical content and consistency of oaths across different institutions, and throughout their education medical students should be encouraged to discuss and reflect on the principles and virtues they will profess when they graduate.

Bacterial promoter repression by DNA looping without protein-protein binding competition.

The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein-protein binding competition. We show that promoters held within such DNA loops are repressed ∼100-fold, with up to an additional ∼10-fold repression (∼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding.

Functional analysis of a de novo ACTB mutation in a patient with atypical Baraitser-Winter syndrome.

Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser-Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals, and we rediagnosed the patient with atypical Baraitser-Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.

Allergic rhinitis.

Allergic rhinitis is a very common disorder that affects people of all ages, peaking in the teenage years. It is frequently ignored, underdiagnosed, misdiagnosed, and mistreated, which not only is detrimental to health but also has societal costs. Although allergic rhinitis is not a serious illness, it is clinically relevant because it underlies many complications, is a major risk factor for poor asthma control, and affects quality of life and productivity at work or school. Management of allergic rhinitis is best when directed by guidelines. A diagnostic trial of a pharmacotherapeutic agent could be started in people with clinically identified allergic rhinitis; however, to confirm the diagnosis, specific IgE reactivity needs to be recorded. Documented IgE reactivity has the added benefit of guiding implementation of environmental controls, which could substantially ameliorate symptoms of allergic rhinitis and might prevent development of asthma, especially in an occupational setting. Many classes of drug are available, effective, and safe. In meta-analyses, intranasal corticosteroids are superior to other treatments, have a good safety profile, and treat all symptoms of allergic rhinitis effectively. First-generation antihistamines are associated with sedation, psychomotor retardation, and reduced academic performance. Only immunotherapy with individually targeted allergens has the potential to alter the natural history of allergic rhinitis. Patients' education is a vital component of treatment. Even with the best pharmacotherapy, one in five affected individuals remains highly symptomatic, and further research is needed in this area.

Overview of the treatment of allergic rhinitis and nonallergic rhinopathy.

Allergic rhinitis (AR) and nonallergic rhinopathy (NAR) represent common nasal conditions affecting millions of individuals across the world. Although patients present with similar symptomatology, those with NAR are frequently affected only after childhood and present with a lack of other comorbid atopic disorders such as asthma, atopic dermatitis, and food allergies. Patients with pure NAR usually have no identifiable specific allergen sensitivity, whereas those with mixed (allergic and nonallergic) rhinitis are sensitized to aeroallergens in a manner that does not fully explain the duration or extent of their symptoms. This review presents the diverse options of currently available pharmacologic agents for the treatment of AR and NAR, including intranasal corticosteroids, H(1)-antihistamines, decongestants, cromolyn sodium, antileukotrienes, anticholinergics, capsaicin, anti-IgE, and intranasal saline, in addition to subcutaneous immunotherapy. Furthermore, treatment algorithms for AR and NAR are presented with a stepped-up, stepped-down scheme to aid the clinician in choosing appropriate therapy.

Pharmacologic rationale for treating allergic and nonallergic rhinitis.

Allergic rhinitis (AR) and perennial nonallergic rhinitis (PNAR) represent conditions affecting millions of individuals across the world. Although the diagnosis of AR might be presumptively based on the types of symptoms and the history of allergen triggers, confirmation requires documentation of specific IgE reactivity. In contrast, PNAR is a condition with similar symptomatology but in which the patient has no identifiable specific allergic sensitivities. This review presents the diverse options of currently available pharmacologic agents for the treatment of AR and PNAR, including intranasal corticosteroids, H1-antihistamines, decongestants, cromolyn sodium, antileukotrienes, anticholinergics, capsaicin, anti-IgE, and intranasal saline. Furthermore, appropriate stepped-up, stepped-down pharmacotherapeutic algorithms are described for the various forms of rhinitis.

Allergic rhinitis: impact of the disease and considerations for management.

AR is a common condition affecting individuals of all ages. Those afflicted with AR often suffer from associated inflammatory conditions of the mucosa,such as AC, rhinosinusitis, asthma, otitis media with effusion, and other atopic conditions, such as eczema and food allergies. Lack of treatment or treatment with suboptimal therapy may result in reduced quality of life and compromise productivity at work or school. Although environmental controls may prove difficult to implement, and not all controls appear adequately to mitigate symptoms of AR, they continue to represent a foundation for treatment. Many different classes of medications are now available, and they have been shown to be effective and safe in a large number of well-designed, double-blind, placebo-controlled clinical trials. Some of the over-the-counter medi-cations have been associated with increased sedation, potentially leading to accidents and fatalities at work or while operating complex machinery, such as automobiles. Only immunotherapy with increasing doses of individually targeted allergens results in sustained changes in the immune system. Although anti-IgE is probably only the first successful immunomodulator commercially available to treat AR, monoclonal antibodies will remain too costly, at least in the near future, to find their way into routine AR treatment.

Association of anti-cyclic citrullinated peptide antibodies, anti-citrullin antibodies, and IgM and IgA rheumatoid factors with serological parameters of disease activity in rheumatoid arthritis.

We evaluated the association of anti-cyclic citrullinated peptide (CCP) antibody titers with serological markers of disease activity. We also compared three different anti-CCP antibody ELISAs with an anti-citrullin ELISA and the IgM and the IgA rheumatoid factor (RF) in their performance of discriminating between rheumatoid arthritis (RA) and other rheumatic diseases. Sera from 333 consecutive patients of the Rheumaeinheit der Medizinischen Poliklinik Munchen, an outpatient clinic for rheumatic diseases, were collected and tested. Anti-CCP antibodies were assayed with three different commercially available ELISAs. Antifilaggrin antibodies were tested with a commercially available ELISA using in vitro deiminated recombinant rat filaggrin. IgA-RF was analyzed with an ELISA, whereas IgM-RF was measured by latex-enhanced turbidimetry. Rheumatoid arthritis (RA) was diagnosed in 87 patients according to the revised classification criteria of the American College of Rheumatology (ACR), probable RA was diagnosed in 23 patients in an early phase not (yet) fulfilling the ACR criteria, and 223 patients had other rheumatic diseases. Differences in sensitivity and specificity were calculated using McNemar's test. A measure of agreement (kappa statistic) was used to examine whether the tests tended to identify the same patients as positive or negative. Correlations between CCP titers and other tests were analyzed by Spearman nonparametric rank correlation. No significant differences in sensitivity and specificity were found between the tested CCP assays (80.0-80.9% and 97.3-98.1%, respectively). All three CCP tests were slightly but not significantly more sensitive and specific than the anti-citrullin assay (77% and 92%, respectively), comparably sensitive but significantly more specific compared with the IgM-RF (86% and 82%, respectively), and significantly more sensitive but comparably specific compared with the IgA-RF (63% and 94.4%, respectively) in detecting the patients with RA. There was no significant correlation between anti-CCP, anti-citrullin, or IgM-RF or IgA-RF antibody titers and C-reactive protein, erythrocyte sedimentation rate, or white blood cell count. A weak but significant linear correlation was found between anti-CCP titers and IgM-RF titers (r = 0.2, P = 0.03). We could not find a significant difference between the three tested anti-CCP assays and the anti-citrullin test in terms of sensitivity and specificity. Compared with the IgM-RF, all the anti-CCP assays were superior in specificity and comparable in sensitivity. Compared with the IgA-RF, they were more sensitive and comparably specific in the discrimination of patients with RA from other rheumatic diseases. No correlation of any tested autoantibody titer with serological parameters of inflammation was found.

Quantum features in atomic nanofabrication using exactly resonant standing waves.

We report on the first fabrication of nanostructures with exactly resonant light revealing the quantum character of the atom-light interaction. Classically the formation of nanostructures is not expected; thus, the observed formation of complex periodic line patterns can be explained only by treating atom-light interaction and propagation of the atoms quantum mechanically. Our numerical quantum calculations are in quantitative agreement with this experimental finding. Moreover, the theory predicts that for small detunings nanostructures with lambda/4 period can be produced, which beats the standard nanofabrication limit of lambda/2. Our experiments confirm this prediction.