Comparing the impact of surgical expert versus non-ophthalmologist instructors on virtual-reality surgical performance: A randomized controlled trial.

PURPOSE: To compare Manual Small Incision Cataract Surgery (MSICS) microsurgical performance in course participants who received virtual reality simulation-based training by either a surgical expert or a non-ophthalmologist instructor. SETTING: Copenhagen Academy for Medical Education and Simulation, Copenhagen, Denmark. DESIGN: Randomized controlled trial. METHODS: Residents and specialists in ophthalmology with no prior MSICS experience were included to receive virtual reality simulation training in MSICS using the HelpMeSee simulator. The participants were randomly allocated to receive training from either an experienced MSICS surgeon or a non-ophthalmologist, also known as near-peer teaching. The performances of the participants were evaluated at baseline and post-training using a MSICS proficiency-based test with evidence of validity. RESULTS: Thirty participants were included in the study and 29 completed the course. There was no significant difference in final test score between the two groups (p = 0.13). The performance score of both groups of participants increased significantly after receiving the training (p < 0.001). All participants passed the proficiency-based test after receiving the training. CONCLUSION: We found no significant difference in surgical proficiency-level whether the participants were trained by a surgical expert or a non-ophthalmologist instructor for MSICS in a virtual-reality based setting. The findings of this study suggest that near-peer teaching within microsurgical performance potentially could be applied with teaching outcomes comparable to a surgical expert-instructor.

A validated test has been developed for assessment of manual small incision cataract surgery skills using virtual reality simulation.

This study investigates the validity evidence of metrics used for the assessment of surgical skills for Manual Small Incision Cataract Surgery (MSICS) in a virtual reality simulator. MSICS surgery is a low-cost, low-technology cataract surgery technique, which is widely used in low- and middle-income countries. However, there is a lack of cataract surgeons globally, and efficient and evidence-based training of new surgeons is needed. In order to investigate the validity of simulator metrics, we included three groups of participants: (1) MSICS novices who were ophthalmologists with no cataract surgery experience, (2) MSICS novices who were experienced phacoemulsification cataract surgeons, but with no MSICS experience, and (3) experienced phacoemulsification and MSICS surgeons. The evaluation included 11 steps of the MSICS procedure, and all simulator metrics for those steps were reviewed. Of the 55 initial metrics, 30 showed high positive discriminative ability. A test passing score of 20 out of 30 was established, and one of 15 novices with no MSICS experience (mean score 15.5) and 7 out of 10 experienced MSICS surgeons (mean score 22.7) passed the test. We have developed and established validity evidence for a test for MSICS skills in a virtual reality simulator for future use in proficiency-based training and evidence-based testing of training interventions.

Predicting the long-term stability of compact multiplanet systems.

We combine analytical understanding of resonant dynamics in two-planet systems with machine-learning techniques to train a model capable of robustly classifying stability in compact multiplanet systems over long timescales of [Formula: see text] orbits. Our Stability of Planetary Orbital Configurations Klassifier (SPOCK) predicts stability using physically motivated summary statistics measured in integrations of the first [Formula: see text] orbits, thus achieving speed-ups of up to [Formula: see text] over full simulations. This computationally opens up the stability-constrained characterization of multiplanet systems. Our model, trained on ∼100,000 three-planet systems sampled at discrete resonances, generalizes both to a sample spanning a continuous period-ratio range, as well as to a large five-planet sample with qualitatively different configurations to our training dataset. Our approach significantly outperforms previous methods based on systems' angular momentum deficit, chaos indicators, and parametrized fits to numerical integrations. We use SPOCK to constrain the free eccentricities between the inner and outer pairs of planets in the Kepler-431 system of three approximately Earth-sized planets to both be below 0.05. Our stability analysis provides significantly stronger eccentricity constraints than currently achievable through either radial velocity or transit-duration measurements for small planets and within a factor of a few of systems that exhibit transit-timing variations (TTVs). Given that current exoplanet-detection strategies now rarely allow for strong TTV constraints [S. Hadden, T. Barclay, M. J. Payne, M. J. Holman, Astrophys. J. 158, 146 (2019)], SPOCK enables a powerful complementary method for precisely characterizing compact multiplanet systems. We publicly release SPOCK for community use.

Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems.

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how "self-avoiding molecular recognition systems" (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer-primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

BACKGROUND: The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. METHODS: We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides). RESULTS: The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels. CONCLUSIONS: Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine 'universal base' and 'mixed sequence' probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.

The mass of the Mars-sized exoplanet Kepler-138 b from transit timing.

Extrasolar planets that pass in front of their host star (transit) cause a temporary decrease in the apparent brightness of the star, providing a direct measure of the planet's size and orbital period. In some systems with multiple transiting planets, the times of the transits are measurably affected by the gravitational interactions between neighbouring planets. In favourable cases, the departures from Keplerian orbits (that is, unaffected by gravitational effects) implied by the observed transit times permit the planetary masses to be measured, which is key to determining their bulk densities. Characterizing rocky planets is particularly difficult, because they are generally smaller and less massive than gaseous planets. Therefore, few exoplanets near the size of Earth have had their masses measured. Here we report the sizes and masses of three planets orbiting Kepler-138, a star much fainter and cooler than the Sun. We determine that the mass of the Mars-sized inner planet, Kepler-138 b, is 0.066(+0.059)(-0.037) Earth masses. Its density is 2.6(+2.4)(-1.5) grams per cubic centimetre. The middle and outer planets are both slightly larger than Earth. The middle planet's density (6.2(+5.8)(-3.4) grams per cubic centimetre) is similar to that of Earth, and the outer planet is less than half as dense at 2.1(+2.2)(-1.2) grams per cubic centimetre, implying that it contains a greater portion of low-density components such as water and hydrogen.

Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research.

Abiotic regioselective phosphorylation of adenosine with borate in formamide.

Nearly 40 years ago, Schoffstall and his coworkers used formamide as a solvent to permit the phosphorylation of nucleosides by inorganic phosphate to give nucleoside phosphates, which (due to their thermodynamic instability with respect to hydrolysis) cannot be easily created in water by an analogous phosphorylation (the "water problem" in prebiotic chemistry). More recently, we showed that borate could stabilize certain carbohydrates against degradation (the "asphalt problem"). Here, we combine the two concepts to show that borate can work in formamide to guide the reactivity of nucleosides under conditions where they are phosphorylated. Specifically, reaction of adenosine in formamide with inorganic phosphate and pyrophosphate in the presence of borate gives adenosine-5'-phosphate as the only detectable phosphorylated product, with formylation (as opposed to hydrolysis) being the competing reaction.

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology.

Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a "single tube" setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a "self-avoiding molecular recognition system" (SAMRS), which enables high levels of multiplexing. The second is an "artificially expanded genetic information system" (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex "liquid microarrays" are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6-10 dengue virions can be detected in a single mosquito.

Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance.

BACKGROUND: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide "letters", the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson-Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. RESULTS: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson-Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2'-deoxyisocytidine and 2'-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. CONCLUSION: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups.

Nucleoside triphosphates having a 3'-ONH2 blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH2 group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH2 blocking group in "next generation sequencing."

Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection.

Any system, natural or human-made, is better understood if we analyze both its history and its structure. Here we combine structural analyses with a "Reconstructed Evolutionary Adaptive Path" (REAP) analysis that used the evolutionary and functional history of DNA polymerases to replace amino acids to enable polymerases to accept a new class of triphosphate substrates, those having their 3'-OH ends blocked as a 3(')-ONH(2) group (dNTP-ONH(2)). Analogous to widely used 2',3'-dideoxynucleoside triphosphates (ddNTPs), dNTP-ONH(2)s terminate primer extension. Unlike ddNTPs, however, primer extension can be resumed by cleaving an O-N bond to restore an -OH group to the 3'-end of the primer. REAP combined with crystallographic analyses identified 35 sites where replacements might improve the ability of Taq to accept dNTP-ONH(2)s. A library of 93 Taq variants, each having replacements at three or four of these sites, held eight variants having improved ability to accept dNTP-ONH(2) substrates. Two of these (A597T, L616A, F667Y, E745H, and E520G, K540I, L616A) performed notably well. The second variant incorporated both dNTP-ONH(2)sand ddNTPs faithfully and efficiently, supporting extension-cleavage-extension cycles applicable in parallel sequencing and in SNP detection through competition between reversible and irreversible terminators. Dissecting these results showed that one replacement (L616A), not previously identified, allows Taq to incorporate both reversible and irreversible terminators. Modeling showed how L616A might open space behind Phe-667, allowing it to move to accommodate the larger 3'-substituent. This work provides polymerases for DNA analyses and shows how evolutionary analyses help explore relationships between structure and function in proteins.

Synthetic biology for improved personalized medicine.

Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than $10,000 (as opposed to $100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and the Westheimer Institute have developed several of these, which will be the topic of this talk..

Incorporation of multiple sequential pseudothymidines by DNA polymerases and their impact on DNA duplex structure.

Thermal denaturation and circular dichroism studies suggested that multiple (up to 12), sequential pseudothymidines, a representative C-glycoside, do not perturb the structure of a representative DNA duplex. Further, various Family A and B DNA polymerases were found to extend a primer by incorporating four sequential pseudothymidine triphosphates, and then continue the extension to generate full-length product. Detailed studies showed that Taq polymerase incorporated up to five sequential C-glycosides, but not more. These results constrain architectures for sequencing, quantitating, and analyzing DNA analogs that exploit C-glycosides, and define better the challenge of creating a synthetic biology using these with natural polymerases.

Synthesis of pyrophosphates for in vitro selection of catalytic RNA molecules.

Reported here are synthetic routes to pyrophosphates linking riboflavin with various nucleosides. The focus is on a flavin-uracil dinucleotide having a biotin tag on the uracil, a molecule that has potential value in the selection of RNA enzymes that catalyze the template-directed polymerization of RNA in the 3'-to-5' direction, which is the direction opposite that catalyzed by standard protein polymerases. Two detailed procedures are presented to prepare this new compound, as well as one procedure to prepare the new flavin-2,6-diaminopurine dinucleotide.

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern.

To support efforts to develop a 'synthetic biology' based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where 'py' indicates a pyrimidine analog and 'pu' indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

Expanding the genetic alphabet: non-epimerizing nucleoside with the pyDDA hydrogen-bonding pattern.

6-Amino-3-(2'-deoxy-beta-D-ribofuranosyl)-5-nitro-1H-pyridin-2-one (4), a C-glycoside exhibiting the nonstandard pyDDA hydrogen-bonding pattern, was synthesized via Heck coupling. The nitro group greatly enhances the stability of the nucleoside toward acid-catalyzed epimerization without leading to significant deprotonation of the heterocycle at physiological pH. These results make nucleoside 4 a promising candidate for an expanded genetic alphabet.

Synthetic biology with artificially expanded genetic information systems. From personalized medicine to extraterrestrial life.

Over 15 years ago, the Benner group noticed that the DNA alphabet need not be limited to the four standard nucleotides known in natural DNA. Rather, twelve nucleobases forming six base pairs joined by mutually exclusive hydrogen bonding patterns are possible within the geometry of the Watson-Crick pair (Fig. 1). Synthesis and studies on these compounds have brought us to the threshold of a synthetic biology, an artificial chemical system that does basic processes needed for life (in particular, Darwinian evolution), but with unnatural chemical structures. At the same time, the artificial genetic information systems (AEGIS) that we have developed have been used in FDA-approved commercial tests for managing HIV and hepatitis C infections in individual patients, and in a tool that seeks the virus for severe acute respiratory syndrome (SARS). AEGIS also supports the next generation of robotic probes to search for genetic molecules on Mars, Europa, and elsewhere where NASA probes will travel.