"Skinny" and "Fat" DNA: Two New Double Helices.

According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.

Ribonucleosides for an artificially expanded genetic information system.

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2'-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson-Crick complement, imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this "second-generation" AEGIS Z:P pair to add amino acids encoded by mRNA.

Electrophysiological variability as marker of dystonia worsening under deep brain stimulation successive withdrawal and renewal effects.

DBS has been shown to be an effective intervention for neurological disorders. However, the intervention is complex and many aspects have not been understood. Various clinical situations have no solution and follow trial and error approaches. Dystonia is a movement disorder characterized by involuntary muscle contractions, which gives rise to abnormal movements and postures. Status dystonicus (SD) represents a life-threatening condition that requires urgent assessment and management. Electrophysiological markers for risk of symptom worsening and SD related patterns of evolution in patients treated with long-term deep brain stimulation (DBS), and specially under the effect of withdrawal and renewals of simulation are needed. To this end, we study the variability of neural synchronization as a mechanism for symptom generation under successive perturbations to a system, i.e. withdrawals and renewals of neuromodulation, through computational simulation of clinical profiles under different plasticity conditions. The simulation shows that the neuroplasticity makeup influences the variability of oscillation synchronization patterns in virtual "patients". The difference between the effect of different electrophysiological signatures is remarkable and under a certain condition (equal medium long term potentiation and long term depression) the situation resembles that of a stable equilibrium, putatively making the sudden worsening or change less likely. Stability of variability can only be observed in this condition and is clearly distinct from other scenarios. CONCLUSION: Our results demonstrate that the neuroplasticity makeup affects the variability of the oscillatory synchrony. This i) informs the shaping of the electrophysiological makeup and ii) might serve as a marker for clinical behavior.

Directed evolution of polymerases to accept nucleotides with nonstandard hydrogen bond patterns.

Artificial genetic systems have been developed by synthetic biologists over the past two decades to include additional nucleotides that form additional nucleobase pairs independent of the standard T:A and C:G pairs. Their use in various tools to detect and analyze DNA and RNA requires polymerases that synthesize duplex DNA containing unnatural base pairs. This is especially true for nested polymerase chain reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard nucleotides, 2-amino-8-(1'-ß-d-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with 6-amino-5-nitro-3-(1'-ß-d-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template. Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed "heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the general rule in directed evolution that "you get what you select for".

Conversion strategy using an expanded genetic alphabet to assay nucleic acids.

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

Reading acquisition reorganizes the phonological awareness network only in alphabetic writing systems.

It is unknown how experience with different types of orthographies influences the neural basis of oral language processing. In order to determine the effects of alphabetic and nonalphabetic writing systems, the current study examined the influence of learning to read on oral language in English and Chinese speakers. Children (8-12 years olds) and adults made rhyming judgments to pairs of spoken words during functional magnetic resonance imaging (fMRI). Developmental increases were seen only for English speakers in the left hemisphere phonological network (superior temporal gyrus (STG), inferior parietal lobule, and inferior frontal gyrus). The increase in the STG was more pronounced for words with conflicting orthography (e.g. pint-mint; jazz-has) even though access to orthography was irrelevant to the task. Moreover, higher reading skill was correlated with greater activation in the STG only for English speaking children. The effects suggest that learning to read reorganizes the phonological awareness network only for alphabetic and not logographic writing systems because of differences in the principles for mapping between orthographic and phonological representations. The reorganization of the auditory cortex may result in better phonological awareness skills in alphabetic readers.

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.

Mechanical Disruption of the Inner Limiting Membrane In Vivo Enhances Targeting to the Inner Retina.

Purpose: To evaluate the effects of mechanical disruption of the inner limiting membrane (ILM) on the ability to target interventions to the inner neurosensory retina in a rodent model. Our study used an animal model to gain insight into the normal physiology of the ILM and advances our understanding of the effects of mechanical ILM removal on the viral transduction of retinal ganglion cells and retinal ganglion cell transplantation. Methods: The ILM in the in vivo rat eye was disrupted using mechanical forces applied to the vitreoretinal interface. Immunohistology and electron microscopy were used to verify the removal of the ILM in retina flatmounts and sections. To assess the degree to which ILM disruption enhanced transvitreal access to the retina, in vivo studies involving intravitreal injections of adeno-associated virus (AAV) to transduce retinal ganglion cells (RGCs) and ex vivo studies involving co-culture of human stem cell-derived RGCs (hRGCs) on retinal explants were performed. RGC transduction efficiency and transplanted hRGC integration with retinal explants were evaluated by immunohistology of the retinas. Results: Mechanical disruption of the ILM in the rodent eye was sufficient to remove the ILM from targeted retinal areas while preserving the underlying retinal nerve fiber layer and RGCs. Removal of the ILM enhanced the transduction efficiency of intravitreally delivered AAV threefold (1380.0 ± 290.1 vs. 442.0 ± 249.3 cells/mm2; N = 6; P = 0.034). Removal of the ILM was also sufficient to promote integration of transplanted RGCs within the inner retina. Conclusions: The ILM is a barrier to transvitreally delivered agents including viral vectors and cells. Mechanical removal of the ILM is sufficient to enhance access to the inner retina, improve viral transduction efficiencies of RGCs, and enhance cellular integration of transplanted RGCs with the retina.

Leadership in Education, Medical Education and Health.

We observe the impact of quality of leadership in our daily lives [...].

Electrophysiological Signature and the Prediction of Deep Brain Stimulation Withdrawal and Insertion Effects.

Deep brain stimulation (DBS) serves as a treatment for neurological and psychiatric disorders, such as Parkinson's disease (PD), essential tremor, dystonia, Tourette Syndrome (GTS), Huntington's disease, and obsessive-compulsive disorder (OCD). There is broad experience with the short-term effects of DBS in individual diseases and their signs/symptoms. However, even in acute treatment and for the same disorder or a given disorder, a prediction of effect is not perfect. Even further, the factors that influence the long-term effect of DBS and its withdrawal are hardly characterized. In this work, we aim to shed light on an important topic, the question of "DBS dependency." To address this, we make use of the Kuramoto model of phase synchronization (oscillation feature) endowed with neuroplasticity to study the effects of DBS under successive withdrawals and renewals of neuromodulation as well as influence of treatment duration in de novo DBS "patients." The results of our simulation show that the characteristics of neuroplasticity have a profound effect on the stability and mutability of oscillation synchronization patterns across successive withdrawal and renewal of DBS in chronic "patients" and also in de novo DBS "patients" with varying duration of treatment (here referred to as the "number of iterations"). Importantly, the results demonstrate the strong effect of the individual neuroplasticity makeup on the behavior of synchrony of oscillatory activity that promotes certain disorder/disease states or symptoms. The effect of DBS-mediated neuromodulation and withdrawal is highly dependent on the makeup of the neuroplastic signature of a disorder or an individual.

2'-deoxy-1-methylpseudocytidine, a stable analog of 2'-deoxy-5-methylisocytidine.

2'-deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2'-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2'-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2'-deoxycytidine and 2'-deoxyguanosine pair.

Hachimoji DNA and RNA: A genetic system with eight building blocks.

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Dynamic assembly of primers on nucleic acid templates.

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of approximately 1 muM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.

Human Depotentiation following Induction of Spike Timing Dependent Plasticity.

Depotentiation (DP) is a crucial mechanism for the tuning of memory traces once LTP (Long Term Potentiation) has been induced via learning, artificial procedures, or other activities. Putative unuseful LTP might be abolished via this process. Its deficiency is thought to play a role in pathologies, such as drug induced dyskinesia. However, since it is thought that it represents a mechanism that is linked to the susceptibility to interference during consolidation of a memory trace, it is an important process to consider when therapeutic interventions, such as psychotherapy, are administered. Perhaps a person with an abnormal depotentiation is prone to lose learned effects very easily or on the other end of the spectrum is prone to overload with previously generated unuseful LTP. Perhaps this process partly explains why some disorders and patients are extremely resistant to therapy. The present study seeks to quantify the relationship between LTP and depotentiation in the human brain by using transcranial magnetic stimulation (TMS) over the cortex of healthy participants. The results provide further evidence that depotentiation can be quantified in humans by use of noninvasive brain stimulation techniques. They provide evidence that a nonfocal rhythmic on its own inefficient stimulation, such as a modified thetaburst stimulation, can depotentiate an associative, focal spike timing-dependent PAS (paired associative stimulation)-induced LTP. Therefore, the depotentiation-like process does not seem to be restricted to specific subgroups of synapses that have undergone LTP before. Most importantly, the induced LTP seems highly correlated with the amount of generated depotentiation in healthy individuals. This might be a phenomenon typical of health and might be distorted in brain pathologies, such as dystonia, or dyskinesias. The ratio of LTP/DP might be a valuable marker for potential distortions of persistence versus deletion of memory traces represented by LTP-like plasticity.

Neural Oscillatory Correlates for Conditioning and Extinction of Fear.

The extinction of conditioned-fear represents a hallmark of current exposure therapies as it has been found to be impaired in people suffering from post-traumatic stress disorder (PTSD) and anxiety. A large body of knowledge focusing on psychophysiological animal and human studies suggests the involvement of key brain structures that interact via neural oscillations during the acquisition and extinction of fear. Consequently, neural oscillatory correlates of such mechanisms appear relevant regarding the development of novel therapeutic approaches to counterbalance abnormal activity in fear-related brain circuits, which, in turn, could alleviate fear and anxiety symptoms. Here, we provide an account of state-of-the-art neural oscillatory correlates for the conditioning and extinction of fear, and also deal with recent translational efforts aimed at fear extinction by neural oscillatory modulation.

Toward a Symptom-Guided Neurostimulation for Gilles de la Tourette Syndrome.

Therapy resistance of approximately one-third of patients with Gilles de la Tourette syndrome (GTS) requires consideration of alternative therapeutic interventions. This article provides a condensed review of the invasive and non-invasive stimulation techniques that have been applied, to date, for treatment and investigation of GTS. Through this perspective and short review, the article discusses potential novel applications for neurostimulation techniques based on a symptom-guided approach. The concept of considering the physiological basis of specific symptoms when using stimulation techniques will provide a platform for more effective non-pharmacological neuromodulation of symptoms in GTS.

Cerebellar neurophysiology in Gilles de la Tourette syndrome and its role as a target for therapeutic intervention.

Therapy resistance of approximately one-third of patients with Gilles de la Tourette syndrome (GTS) requires consideration of alternative therapeutic interventions. The article demonstrates the role of the cerebellum in neuropsychiatric disorders and GTS in particular, specifically its role in functions relating to motor and cognitive symptoms. Certain circuits in the cerebellum have been shown to undergo learning-induced changes during conditioning, with cells in the cortex of the cerebellum appearing to decrease their activity whilst those in deep nuclei seem to do the inverse. Evidence exists showing that abnormal excitability of the motor cortex via the cerebellum could be expected to participate in motor tics in GTS possibly due to aberrations in certain structures of involved circuits. The role of the cerebellum in learning and plasticity processes renders it a strategic and valuable structure to consider for brain stimulation when investigating potential treatment options for neuropsychiatric disorders such as GTS. This article puts forth the concept of using non-invasive and invasive brain stimulation techniques as a novel platform for non-pharmacological neuromodulation of GTS symptoms.

Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System.

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ∼92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ∼86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.