Παρασκευή, 27 Απριλίου 2018

A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast

The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.



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Ancestral State Reconstruction of the Apoptosis Machinery in the Common Ancestor of Eukaryotes

Apoptotic cell death is a type of eukaryotic cell death. In animals, it regulates development, is involved in cancer suppression, and causes cell death during pathological aging of neuronal cells in neurodegenerative diseases such as Alzheimer's. Mitochondrial apoptotic-like cell death, a form of primordial apoptosis, also occurs in unicellular organisms. Here, we ask the question why the apoptosis machinery has been acquired and maintained in unicellular organisms and attempt to answer it by performing ancestral state reconstruction. We found indications of an ancient evolutionary arms race between protomitochondria and host cells, leading to the establishment of the currently existing apoptotic pathways. According to this reconstruction, the ancestral protomitochondrial apoptosis machinery contained both caspases and metacaspases, four types of apoptosis induction factors (AIFs), both fungal and animal OMI/HTR proteases, and various apoptotic DNases. This leads to the prediction that in extant unicellular eukaryotes, the apoptotic factors are involved in mitochondrial respiration and their activity is needed exclusively in aerobic conditions. We test this prediction experimentally using yeast and find that a loss of the main apoptotic factors is beneficial under anaerobic conditions yet deleterious under aerobic conditions in the absence of lethal stimuli. We also point out potential medical implications of these findings.



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A High-Quality Reference Genome for the Invasive Mosquitofish Gambusia affinis Using a Chicago Library

The western mosquitofish, Gambusia affinis, is a freshwater poecilid fish native to the southeastern United States but with a global distribution due to widespread human introduction. Gambusia affinis has been used as a model species for a broad range of evolutionary and ecological studies. We sequenced the genome of a male G. affinis to facilitate genetic studies in diverse fields including invasion biology and comparative genetics. We generated Illumina short read data from paired-end libraries and in vitro proximity-ligation libraries. We obtained 54.9x coverage, N50 contig length of 17.6 kb, and N50 scaffold length of 6.65 Mb. Compared to two other species in the Poeciliidae family, G. affinis has slightly fewer genes that have shorter total, exon, and intron length on average. Using a set of universal single-copy orthologs in fish genomes, we found 95.5% of these genes were complete in the G. affinis assembly. The number of transposable elements in the G. affinis assembly is similar to those of closely related species. The high-quality genome sequence and annotations we report will be valuable resources for scientists to map the genetic architecture of traits of interest in this species.



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Is the behavior of disc replacement adjacent to fusion affected by the location of the fused level in hybrid surgery?

Context: Hybrid surgery (HS), consisting of cervical disc arthroplasty (CDA) at the mobile level, along with anterior cervical discectomy and fusion (ACDF) at the spondylotic level, could be a promising treatment for patients with multilevel cervical degenerative disc disease (DDD). An advantage of this technique is that it utilizes an optimal procedure according to the status of each level. However, information is lacking regarding the influence of the relative location of the replacement and the fusion segment in vivo.

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How social media, training, and demographics influence online reviews across three leading review websites for spine surgeons

The future of healthcare is consumer driven with a focus on outcome metrics and patient feedback. Physician review websites have grown in popularity and are guiding patients to certain healthcare providers, for better or worse. No prior study has specifically evaluated internet reviews of spine surgeons, determined if social media correlates with patient reviews, or evaluated Google as a physician review website.

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Use of the subcutaneous lumbar spine (SLS) index as a predictor for surgical complications in lumbar spine surgery

Lumbar spine surgeries require adequate exposure to visualize key structures and limited exposure can make surgery more technically difficult, thus increasing the potential for complications. Body mass index and body mass distribution have been shown to be associated with worse surgical outcomes.

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Predictive model for distal junctional kyphosis after cervical deformity surgery

Distal Junctional Kyphosis (DJK) is a primary concern of surgeons correcting cervical deformity. Identifying patients and procedures at higher risk for developing this condition is paramount in improving patient selection and care.

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Decompression surgery improves gait quality in patients with symptomatic lumbar spinal stenosis

To fully understand the extent of limitations associated with symptomatic lumbar spinal stenosis (LSS) and the functional outcome of its treatment, not only function during daily activities (e.g. using the 6-minute walk test (6MWT)) but also the quality of function should be objectively assessed.

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Chronic pre-operative opioid use is a risk factor for increased complications, resource use and costs after cervical fusion

As healthcare transitions to value-based models, there has been an increased focus on patient factors that can influence peri- and post-operative adverse events, resource use, and costs. Many studies have reported risk factors for systemic complications after cervical fusion, but none have studied chronic opioid therapy (COT) as a risk factor.

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The META score for differentiating metastatic from osteoporotic vertebral fractures: an independent agreement assessment

Differentiating osteoporotic vertebral fractures (OVF) from metastatic vertebral fractures (MVF) is an important clinical challenge. A novel magnetic resonance imaging- (MRI) based score (the META score) was described, aiming to differentiate OVF and MVF. This score showed an almost perfect agreement by the group developing it, but an independent agreement evaluation is pending.

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Increase in chromogranin A- and serotonin-positive cells in pouch mucosa of patients with ulcerative colitis undergoing proctocolectomy

Inflammatory bowel disease (IBD) is associated with neuroendocrine cell hyperplasia.

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Peroxisome Proliferator-activated Receptor Gamma Activation Promotes Intestinal Barrier Function by Improving Mucus and Tight Junctions in a Mouse Colitis Model

Defects in mucus and intestinal epithelia can lead to intestinal inflammation in colitis. Reduced peroxisome proliferator-activated receptor gamma (PPARγ) in the mucosa may contribute to inflammation. However, the roles of PPARγ in the intestinal barrier remain poorly understood.

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New concepts on intestinal microbiota and the role of the non-absorbable antibiotics with special reference to rifaximin in digestive diseases

Digestive diseases are a broad range of chronic disorders that substantially and negatively impact the patients' quality of life. Here, we review our current understanding on the pathophysiology of hepatic encephalopathy, irritable bowel syndrome, and diverticular disease, with a special focus on the gut microbiota composition associated with these disorders. Furthermore, we review the current clinical practise for their therapeutic treatments, including probiotics, diet change, non-adsorbable disaccharides, and antibiotics.

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Endoscopic management of a colonic fistula communicating with a walled-off pancreatic necrosis



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Quality and Competence in Endoscopic Retrograde Cholangiopancreatography − where are we 50 years later?

Training in endoscopic retrograde cholangiopancreatography (ERCP) requires the development of technical, cognitive, and integrative skills well beyond those needed for standard endoscopic procedures. So far, there are limited data regarding what constitutes competency in ERCP, including achievement and maintenance. Recent studies have highlighted overall procedural numbers are not enough to warrant competency, although more is better. We performed a comprehensive literature search until June 2017 using predetermined search terms to identify relevant articles and summarized their results as a narrative review.

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Side-by-side stent deployment above the papilla to treat hepatic hilar obstruction using a new covered metal stent



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Evaluation of T cells in blood after a short gluten challenge for coeliac disease diagnosis

To diagnose coeliac disease (CD) in individuals on a gluten free diet (GFD), we aimed to assess the utility of detecting activated γd and CD8 T cells expressing gut-homing receptors after a short gluten challenge.

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Does cyst growth predict malignancy in branch duct intraductal papillary mucinous neoplasms? Results of a large multicenter experience

Cyst growth of BD-IPMNs on follow-up imaging remains a concerning sign.

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Annual NAVIGATOR conference highlights achievements of two outstanding emergency telecommunicators

Brought to you by Priority Dispatch LAS VEGAS — The International Academies of Emergency Dispatch® (IAED™) has recognized the outstanding accomplishments of emergency dispatchers and the contributions of dedicated professionals at its annual NAVIGATOR conference, held April 24–26 at the Aria Resort and Casino in Las Vegas, Nevada. At the event, IAED officials presented awards to ...

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Pulsara named one of Montana High Tech Business Alliance’s 12 High Growth companies to watch in 2018

BOZEMAN, Mont. — The spotlight is being shone once again on Pulsara, recently named one of Montana High Tech Business Alliance's 12 High Growth companies to watch in 2018. Pulsara is excited to be recognized as one of Montana's rising stars among over 500 high-tech companies in the state. Pulsara is a healthcare product that securely enables all members of a patient care team — ...

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A German‐Jewish refugee in Vichy France 1939–1941. Arno Motulsky's memoir of life in the internment camps at St. Cyprien and Gurs

American Journal of Medical Genetics Part A, EarlyView.


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The phenotypic spectrum of Xia‐Gibbs syndrome

American Journal of Medical Genetics Part A, EarlyView.


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Functional independence of Taiwanese children with Prader–Willi syndrome

American Journal of Medical Genetics Part A, EarlyView.


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Neurodevelopmental outcome in 22q11.2 deletion syndrome and management

American Journal of Medical Genetics Part A, EarlyView.


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38th Annual David W. Smith Workshop on Malformations and Morphogenesis: Abstracts of the 2017 Annual Meeting

American Journal of Medical Genetics Part A, EarlyView.


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The ontogeny of Robin sequence

American Journal of Medical Genetics Part A, EarlyView.


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X‐linked intellectual disability update 2017

American Journal of Medical Genetics Part A, EarlyView.


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Delayed peak response of cortisol to insulin tolerance test in patients with Prader–Willi syndrome

American Journal of Medical Genetics Part A, EarlyView.


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KBG syndrome patient due to 16q24.3 microdeletion presenting with a paratesticular rhabdoid tumor: Coincidence or cancer predisposition?

American Journal of Medical Genetics Part A, EarlyView.


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Mutations in SZT2 result in early‐onset epileptic encephalopathy and leukoencephalopathy

American Journal of Medical Genetics Part A, EarlyView.


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Expanding the phenotype of congenital central hypoventilation syndrome impacts management decisions

American Journal of Medical Genetics Part A, EarlyView.


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An assessment of health, social, communication, and daily living skills of adults with down syndrome

American Journal of Medical Genetics Part A, EarlyView.


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Association of 17q24.2‐q24.3 deletions with recognizable phenotype and short telomeres

American Journal of Medical Genetics Part A, EarlyView.


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Arno G. Motulsky, 1923–2018, Luck and Service

American Journal of Medical Genetics Part A, EarlyView.


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Correction to: Evaluation of pharmacokinetic models of intravenous dexmedetomidine in sedated patients under spinal anesthesia

Inadvertently, the reference [8] was published incorrectly in the original publication of the article. The correct reference [8] is provided below:



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The impact of general anesthesia on child development and school performance: a population‐based study

Pediatric Anesthesia, EarlyView.


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Consensus statement on clear fluids fasting for elective pediatirc general anesthesia

Pediatric Anesthesia, EarlyView.


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