INTRODUCTION It has long been an interesting question whether a living cell can be constructed from scratch in the lab, a goal that may not be realized anytime soon. Nonetheless, with advances in DNA synthesis technology, the complete genetic material of an organism can now be synthesized chemically. Hitherto, genomes of several organisms including viruses, phages, and bacteria have been designed and constructed. These synthetic genomes are able to direct all normal biological functions, capable of self-replication and production of offspring. Several years ago, a group of scientists worldwide formed an international consortium to reconstruct the genome of budding yeast, Saccharomyces cerevisiae . RATIONALE The synthetic yeast genome, designated Sc2.0, was designed according to a set of arbitrary rules, including the elimination of transposable elements and incorporation of specific DNA elements to facilitate further genome manipulation. Among the 16 S. cerevisiae chromosomes, chromosome XII is unique as one of the longest yeast chromosomes (~1 million base pairs) and additionally encodes the highly repetitive ribosomal DNA locus, which forms the well-organized nucleolus. We report on the design, construction, and characterization of chromosome XII, the physically largest chromosome in S. cerevisiae. RESULTS A 976,067–base pair linear chromosome, synXII, was designed based on the native chromosome XII sequence of S. cerevisiae , and chemically synthesized. SynXII was assembled using a two-step method involving, successive megachunk integration to produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding a full-length functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII were caused by deletion of tRNA genes and were corrected by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit. The same synthetic rDNA unit was also used to regenerate rDNA at three distinct chromosomal locations. The rDNA signature sequences of the internal transcribed spacer (ITS), often used to determine species identity by standard DNA barcoding procedures, were swapped to generate a Saccharomyces synXII strain that would be identified as S. bayanus. Remarkably, these substantial DNA changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION The rDNA locus of synXII is highly plastic; not only can it be moved to other chromosomal loci, it can also be altered in its ITS region to masquerade as a distinct species as defined by DNA barcoding, used widely in taxonomy. The ability to perform “species morphing” reported here presumably reflects the degree of evolutionary flexibility by which these ITS regions change. However, this barcoding region is clearly not infinitely flexible, as only relatively modest intragenus base changes were tolerated. More severe intergenus differences in ITS sequence did not result in functional rDNAs, probably because of defects in rRNA processing. The ability to design, build, and debug a megabase-sized chromosome, together with the flexibility in rDNA locus position, speaks to the remarkable overall flexibility of the yeast genome. Hierarchical assembly and subsequent restructuring of synXII. SynXII was assembled in two steps: First, six semisynthetic synXII strains were built in which segments of native XII DNA were replaced with the corresponding designer sequences. Next, the semisynthetic strains were combined withmultiple rounds ofmating/sporulation, eventually generating a single strain encoding fulllength synXII.The rDNA repeats were removed, modified, and subsequently regenerated at distinct chromosomal locations for species morphing and genome restructuring.

INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain. CONCLUSION Synthet...

Objective: Ultra-high-field 7-Tesla (7T) magnetic resonance imaging (MRI) offers increased signal-to-noise and contrast-to-noise ratios, which may improve visualization of cortical malformations. We aim to assess the clinical value of in vivo structural 7T MRI and its post-processing for the noninvasive identification of epileptic brain lesions in patients with pharmacoresistant epilepsy and nonlesional 3T MRI who are undergoing presurgical evaluation. Methods: Sixty-seven patients were included who had nonlesional 3T MRI by official radiology report. Epilepsy protocols were used for the 3T and 7T acquisitions. Post-processing of the 7T T1-weighted magnetization-prepared two rapid acquisition gradient echoes sequence was performed using the morphometric analysis program (MAP) with comparison to a normal database consisting of 50 healthy controls. Review of 7T was performed by an experienced board-certified neuroradiologist and at the multimodal patient management conference. The clinical significance of 7T findings was assessed based on intracranial electroencephalography (ICEEG) ictal onset, surgery, postoperative seizure outcomes, and histopathology. Results: Unaided visual review of 7T detected previously unappreciated subtle lesions in 22% (15/67). When aided by 7T MAP, the total yield increased to 43% (29/67). The location of the 7T-identified lesion was identical to or contained within the ICEEG ictal onset in 13 of 16 (81%). Complete resection of the 7T-identified lesion was associated with seizure freedom (P = .03). Histopathology of the 7T-identified lesions encountered mainly focal cortical dysplasia (FCD). 7T MAP yielded 25% more lesions (6/24) than 3T MAP, and showed improved conspicuity in 46% (11/24). Significance: Our data suggest a major benefit of 7T with post-processing for detecting subtle FCD lesions for patients with pharmacoresistant epilepsy and nonlesional 3T MRI.

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Background and purpose Pre‐surgical evaluation of pediatric patients with drug‐resistant focal epilepsy and negative (non‐lesional) magnetic resonance imaging (MRI) is particularly challenging. Focal cortical dysplasia (FCD), a frequent pathological substrate in such setting, may be subtle on MRI and evade detection. The aim of this study was to use voxel‐based MRI postprocessing to improve the detection of subtle FCD in pediatric surgical candidates. Methods A consecutive cohort of pediatric patients undergoing pre‐surgical evaluation with a negative MRI by visual analysis was included. MRI postprocessing was performed using a voxel‐based morphometric analysis program (MAP) on T1‐weighted volumetric MRI, with comparison to an age‐specific normal pediatric database. The pertinence of MAP‐positive areas was confirmed by surgical outcome and pathology. Results A total of 78 patients were included. Forty‐four patients (56%) had positive MAP regions. Complete resection of the MAP‐positive regions was positively associated with seizure‐free outcome compared with the no/partial resection group (P < 0.001). Patients with no/partial resection of the MAP‐positive regions had worse seizure outcomes than the MAP‐negative group (P = 0.002). The MAP‐positive rate was 100%, 77%, 63% and 40% in the 3–5, 5–10, 10–15 and 15–21 year age groups, respectively. MAP‐positive rates were 45% in patients with temporal resection and 63% in patients with extratemporal resection. Complete resection of the MAP‐positive regions was positively associated with seizure‐free outcome in the extratemporal group (P = 0.001) but not in the temporal group (P = 0.070). Conclusion Our data suggest the importance of using MRI postprocessing in the pre‐surgical evaluation process of pediatric epilepsy patients with apparently normal MRI.

SummaryThe revolution in theory, swift technological developments, and invention of new devices have driven tremendous progress in neurostimulation as a third‐line treatment for epilepsy. Over the past decades, neurostimulation took its place in the field of epilepsy as an advanced treatment technique and opened up a new world. Numerous animal studies have proven the physical efficacy of stimulation of the brain and peripheral nerves. Based on this optimistic fundamental research, new advanced techniques are being explored in clinical practice. Over the past century, drawing on the benefits brought about by vagus nerve stimulation for the treatment of epilepsy, various new neurostimulation modalities have been developed to control seizures. Clinical studies including case reports, case series, and clinical trials have been booming in the past several years. This article gives a comprehensive review of most of these clinical studies. In addition to highlighting the advantages of neurostimulation for the treatment of epilepsy, concerns with this modality and future development directions are also discussed. The biggest advantage of neurostimulation over pharmacological treatments for epilepsy is the modulation of the epilepsy network by delivering stimuli at a specific target or the “hub.” Conversely, however, a lack of knowledge of epilepsy networks and the mechanisms of neurostimulation may hinder further development. Therefore, theoretical research on the mechanism of epileptogenesis and epilepsy networks is needed in the future. Within the multiple modalities of neuromodulation, the final choice should be made after full discussion with a multidisciplinary team at a presurgical conference. Furthermore, the establishment of a neurostimulation system with standardized parameters and rigorous guidelines is another important issue. To achieve this goal, a worldwide collaboration of epilepsy centers is also suggested in the future.

The interaction between dorsal and ventral attention networks (VANs) is mediated by the middle frontal gyrus (MFG), which is functionally connected to both networks. However, the direct role of the MFG in selective and sustained attention remains controversial. In the current study, we used transcranial magnetic stimulation (TMS) and electroencephalography (EEG) to probe the connectivity dynamic changes of MFG-associated regions during different attention modes. The participants underwent visual, selective, and sustained attention tasks to observe TMS-induced network changes. Twenty healthy participants received single-pulse TMS over the left or right MFG during tasks, while synchronous EEG data was acquired. Behavioral results were recorded and time-varying brain network analyses were performed. We found that the MFG is involved in attention processing and that sustained attention was preferentially controlled by the right MFG. Moreover, compared with the right hemisphere, the left hemisphere was associated with selective attention tasks. Visual and selective attention tasks induced MFG-related changes in network nodes were within the left hemisphere; however, sustained attention induced changes in network nodes were in the bilateral posterior MFG. Our findings indicated that the MFG plays a crucial role in regulating attention networks. In particular, TMS-induced MFG alterations influenced key nodes of the time-varying brain network, leading to the reorganization of brain network modules.

Background: Epileptic spasms (ES) is a severe seizure type and lack of adequate methods for controlling of clinical attacks. Previous studies have indicated that cathodal transcranial direct current stimulation (tDCS) reduces seizure frequency for patients with epilepsy. ES are proposed to have a focal cortical origin. We hypothesized that patients with ES exhibit hyperactive network hubs in the parietal lobe, and that cathodal tDCS targeting the bilateral parietal region can reduce seizure frequency in patients with pharmacoresistant ES.Materials and Methods: The present study consisted of three basic phases: (a) a pre-treatment monitoring period for 14 days; (b) a consecutive 14-day treatment period during which patients were treated with 1 or 2 mA cathode tDCS for 40 min once per day; (c) and a follow-up period for at least 28 days. During the first 20 min of treatment, the cathode was placed over the right parietal lobe (P4) with the reference electrode over the contralateral supra-orbital area. In the second 20 min, the cathode was placed over the left parietal lobe (P3), with the reference electrode over the contralateral supra-orbital area. All patients received active tDCS treatment, and some patients underwent more than one treatment block. Patients maintained a seizure diary throughout the study. Antiepileptic drug therapy remained unchanged throughout the study. K-related samples Friedman tests and two-related samples tests were used to analyze data from all patients.Results: Seven patients with pharmacoresistant ES were included, receiving a total of eighteen 14-day blocks of tDCS treatment. We observed a significant difference in seizure frequency at the second month (p = 0.028, unadjusted), as well as a trend toward decreased seizure frequency at the fourth month (p = 0.068, unadjusted) of the first follow-up, relative to baseline. Three of seven patients (42.9%) exhibited sustained seizure reduction, while one (14.3%) experienced a short-term reduction in seizure frequency following cathodal tDCS treatment. Treatment was well tolerated in all patients.Conclusions: Repeated tDCS with the cathode placed over the bilateral parietal region is safe and may be effective for reducing seizure frequency in a subgroup of patients with pharmacoresistant ES.