bluuebunny/crossref_metadata_2025 huggingface dataset:
VS01.07: THORACOSCOPIC ESOPHAGECTOMY WITH A HYBRID OF THE PRONE AND LEFT LATERAL DECUBITUS POSITIONS FOR ESOPHAGEAL CARCINOMA
Authors: Kawakubo Hirofumi , Mayanagi Shuhei , Kitagawa Yuko
Published: September 2018

Description

Because esophagectomy with radical lymphadenectomy is highly invasive, thoracoscopic esophagectomy (TE) is attracting attention as a less invasive procedure. We first performed TE with the left decubitus position in 1996. In 2009 we developed a hybrid of the prone and left lateral decubitus positions for TE, and a total of 420 patients underwent TE with a hybrid position. We introduced TE with a hybrid position for the following three reasons: (1) Mobilization and lymphadenectomy around the middle and lower esophagus are easier in the prone position. Thanks to artificial pneumothorax and the gravity, the middle and lower mediastinum are opened, and which give us good surgical field. (2) Lymphadenectomy along the left recurrent laryngeal nerve (RLN) is more reliable and precise when performed in the left lateral decubitus position. We can dissect lymph node around the RLN higher position in the upper mediastinum. (3) Unexpected events requiring conversion to thoracotomy (e.g. massive bleeding, injury of other organs, dense intrathoracic adhesion, resection of adjacent organs) are easier to deal with in the left lateral decubitus position. The patient is fixed on the operating table with the semi-prone position and we can easily change patient positions from the left lateral decubitus position to the prone position and vice versa using rotation system of the operation table. The upper mediastinal procedure including lymphadenectomy along the right and left RLN is performed with the patient in the left lateral decubitus position, while the middle and lower mediastinal procedures are performed with the patient in the prone position with artificial pneumothorax (7mmHg). The abdominal procedures have beenwere performed by hand-assisted laparoscopic surgery (HALS) and gastric tube reconstruction in thethrough a posterior mediastinal route was performed as s a standard surgical procedure in our institution. The magnifying effect of thoracoscope enables us to perform more precise surgery and preserve nerve and vessels, and a hybrid position is thought to be feasible and effective methods.

Disclosure

All authors have declared no conflicts of interest.
VS01.08: STEPWISE APPROACH FOR ROBOT-ASSISTED THORACOSCOPIC LEFT RECURRENT LARYNGEAL NERVE LYMPH NODE DISSECTION
Authors: Chao Yin-Kai
Published: September 2018

Description

This video showed the stepwise approach for robot-assisted thoracoscopic left recurrent laryngeal nerve lymph node dissection. With the patient lying in the left semiprone position, a total of five trocars were positioned. Left RLN LND was begun by releasing the right esophagotracheal band. Subsequently, the trachea was gradually rotated clockwise through the application of a gentle pressure on the left edge of the tracheal cartilage with the goal of exposing and releasing the left esophagotracheal band. After the complete release of the bilateral esophagotracheal band, the esophagus was retracted toward the dorsal side with the right first robotic arm. Simultaneously, an assistant rolled back the trachea using a forceps grasping a small piece of gauze, with the aim of improving operative exposure. The left RLN was identified in the middle of the soft tissue between the trachea and the esophagus. Dissection was started from the ventral side RLN nodes and subsequently continued toward the dorsal side until the total skeletonization of the left RLN

Disclosure

All authors have declared no conflicts of interest.
VS02.09: LAPAROSCOPIC REPAIR OF RECURRENT TYPE IV PARAESOPHAGEAL HERNIA WITH COLLIS GASTROPLASTY
Authors: Hughes Timothy , Sclabas Guido , Chow Geoffrey
Published: September 2018

Description

Laparoscopic Repair of Recurrent Type IV Hiatal Hernia with Collis Gastroplasty In this case, we present a laparoscopic repair of a large, recurrent type four paraesophageal hernia with a Collis Gastroplasty. The patient presented 4 months after repair of an initial hernia at a separate facility with primary hiatal closure without an anti-reflux procedure. Intra-operatively, there was a giant type IV hernia with the transverse colon and the majority of the stomach in the right chest with an organ-oaxial volvulus. The colon and omentum were easily reducible, but the stomach was densely adherent within the right chest. After extensive dissection, the stomach was able to be reduced into the abdomen, but only two centimeters of intra-abdominal esophagus remained and a Collis gastroplasty was performed. A Nissen fundoplication was then performed at the end of the case. This case demonstrates three unique findings. The first is the short time to recurrence after a prior repair without any establishment of an anti-reflux barrier. The second is the unusual hernia with the majority of the stomach located in the right chest. Finally, this video demonstrates the technique of a laparoscopic Collis gastroplasty with a wedge fundectomy technique given the intra-operative findings of a short esophagus. This technique is a reproducible approach that creates a neo-esophagus that can be considered when there is axial tension after full mobilization of the esophagus. Identification of optimal location for stapler placement is facilitated by concurrent laparoscopy with endoscopic identification of the gastroesophageal junction.

Disclosure

All authors have declared no conflicts of interest.
VS03.04: MINIMALLY INVASIVE IVOR LEWIS ESOPHAGECTOMY WITH ROBOTIC ASSISTANCE AND IMMUNOFLUORESCENCE: MODULAR STEP UP APPROACH FOR SAFE INTRODUCTION OF NEW TECHNOLOGY
Authors: Fuchs Hans , Lambertz Rolf , Schröder Wolfgang , Leers Jessica , Bruns Christiane
Published: September 2018

Description

Minimally invasive technologies have improved outcomes after esophagectomy and the use of robotic technology in Europe is rapidly increasing. Aim of this study is to evaluate the introduction of new technologies in a center of excellence for upper gastrointestinal surgery.

Methods

A standardized teaching protocol of a complete OR team was performed in simulation and animal models at the center for the future of surgery (San Diego, CA) and IRCAD (Strasbourg, France) to receive certification as console surgeons. Starting 02/2017 the davinci xi and stryker ICG laparoscopy systems were introduced at our academic center (certified center of excellence for surgery of the upper gastrointestinal tract, n > 300 esophageal cases/year). After simple training procedures based on our minimally invasive expertise were performed, difficulty was increased based on a modular step up approach to safely perform robotic thoracic assisted Ivor Lewis esophagectomy.

Results

From 02/2017–02/2018, a total of 35 robotic cases were performed. All cases were performed safely without operation-associated complications. Level of difficulty was increased based on our modular step up approach without quality compromises. Video documentation using the new technology is provided.

Conclusion

The standardized training protocol and our modular step up approach allowed safe introduction of the new technology used. All cases were performed safely without operation-associated complications.

Disclosure

All authors have declared no conflicts of interest.
VS03.09: PRONE POSITION THORACOSCOPIC ESOPHAGECTOMY WITH INTRATHORACIC ESOPHAGOGASTRIC ANASTOMOSIS
Authors: Sancho-Muriel Jorge , Mingol Fernando , Nuñez Ruth , Vaqué Javier , De Jesús Milton , Cholewa Hanna , García Rafael , García-Granero Eduardo
Published: September 2018

Description

New minimally invasive techniques in esophageal surgery have shown promising results in postoperative care, reducing morbidity such as postoperative pain, respiratory complications due to thoracotomy and length of hospital stay among others, especially by the use of a thoracoscopic approach in the prone position. One of the reasons that limits the use of this technique the most is the intrathoracic thoracoscopic anastomosis, which is a very demanding procedure and the failure of which implicates high mortality. This video presents a thoracoscopic Ivor-Lewis esophagectomy in prone position, without selective intubation. We perform an en bloc esophagectomy with the resection of both pleuras and all of the lymphatic and adipose tissue around the esophagus up to the aortic plane, with an additional infracarinal lymphadenectomy. The subsequent reconstruction is an intrathoracic esophagogastric anastomosis using an Orvil® device and a manual tobacco-pouch suture to fit the head of the circular endostapler. This suture is reinforced by a laparoscopic endoloop. To perform this technique it is mandatory to make an assistance minithoracotomy for the placement of a gelport that allows the surgical specimen extraction and the placement of the circular endostapler without losing the pneumothorax, crucial for the surgical intervention. The aim of this video is to present step by step the thoracoscopic part of a minimally invasive, Ivor-Lewis subtotal esophagectomy, using the Orvil® device to perform the intrathoracic anastomosis.

Disclosure

All authors have declared no conflicts of interest.
Embryonic Stem Cell–Derived mmu-miR-291a-3p Inhibits Cellular Senescence in Human Dermal Fibroblasts Through the TGF-β Receptor 2 Pathway
Authors: Bae Yun-Ui , Son Youlim , Kim Chang-Hyun , Kim Kwang Seok , Hyun Se Hee , Woo Hyun Goo , Jee Byul A , Choi Jun-Hyuk , Sung Hoon-Ki , Choi Hyung-Chul , Park So Young , Bae Ju-Hyun , Doh Kyung-Oh , Kim Jae-Ryong
Published: September 2018

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation–induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated β-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-β receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-β receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases.
Critique and Alternativity in International Relations
Authors: Visoka Gëzim
Published: September 2018

This article critically interrogates the episteme of alternativity in international relations (IR) to rethink the purpose of critical knowledge in global politics. It questions what critical knowledge is for and whose purpose it serves. While alternativity is the very condition that has given rise to critical approaches, there is a deep-rooted division among critical scholars regarding the relationship between criticality and alternativity. This article argues that alternativity provides an opportunity for critical scholars to remain relevant without being affiliated with positivist logics of inquiry. In examining the potential of alternativity, the article explores three modes of alternativity in peace and conflict studies: critique-without-alternative, critique-as-alternative, and critique-with-alternative. It probes the merits and limits of the episteme of alternativity in generating new possibilities for advancing emancipatory interests and saving critical theory from losing its original transformative impetus. In the final part, the article explores future directions for rejuvenating the purpose of critique by exploring the nexus between criticality and alternativity on postparadigmatic and practical grounds.
Spatial Chromatin Architecture Alteration by Structural Variations in Human Genomes at Population Scale
Authors: Sadowski Michal , Kraft Agnieszka , Szalaj Przemyslaw , Wlasnowolski Michal , Tang Zhonghui , Ruan Yijun , Plewczynski Dariusz
Published: September 2018

This genome-wide study is focused on the impact of structural variants identified in individuals from 26 human populations onto three-dimensional structures of their genomes. We assess the tendency of structural variants to accumulate in spatially interacting genomic segments and design a high-resolution computational algorithm to model the 3D conformational changes resulted by structural variations. We show that differential gene transcription is closely linked to variation in chromatin interaction networks mediated by RNA polymerase II. We also demonstrate that CTCF-mediated interactions are well conserved across population, but enriched with disease-associated SNPs. Altogether, this study assesses the critical impact of structural variants on the higher order organization of chromatin folding and provides unique insight into the mechanisms regulating gene transcription at the population scale, among which the local arrangement of chromatin loops seems to be the leading one. It is the first insight into the variability of the human 3D genome at the population scale.
Stage specific classification of DEGs via statistical profiling and network analysis reveals potential biomarker associated with various stages of TB
Authors: Ishrat Romana
Published: September 2018

Background

Tuberculosis (TB) is a deadly transmissible disease that can infect almost any body-part of the host but is mostly infect the lungs. It is one of the top 10 causes of death worldwide. In the 30 high TB burden countries, 87% of new TB cases occurred in 2016. Seven countries: India, Indonesia, China, Philippines, Pakistan, Nigeria, and South Africa accounted for 64% of the new TB cases. To stop the infection and progression of the disease, early detection of TB is important. In our study, we used microarray data set and compared the gene expression profiles obtained from blood samples of patients with different datasets of Healthy control, Latent infection, Active TB and performed network-based analysis of DEGs to identify potential biomarker.

Objectives

We want to observe the transition of genes from normal condition to different stages of the TB and identify, annotate those genes/pathways/processes that play key role in the progression of TB disease during its cyclic interventions in human body.

Results

We identified 319 genes that are differentially expressed in various stages of TB (Normal to LTTB, Normal to Active TB and LTTB to active TB) and allocated to pathways from multiple databases which comprised of curated class of associated genes. These pathway’s importance was then evaluated according to the no. of DEGs present in the pathway and these genes show the broad spectrum of processes that take part in every state. In addition, we studied the regulatory networks of these classified genes, network analysis does consider the interactions between genes (specific for TB) or proteins provide us new facts about TB disease, which in turn can be used for potential biomarkers identification. We identified total 29 biomarkers from various comparison groups of TB stages in which 14 genes are over expressed as host responses against pathogen, but 15 genes are down regulated that means these genes has allowed the process of host defense to cease and give time to pathogen for its progression.

Conclusions

This study revealed that gene-expression profiles can be used to identify and classified the genes on stage specific pattern among normal, LTTB and active TB and network modules associated with various stages of TB were elucidated, which in turn provided a basis for the identification of potential pathways and key regulatory genes that may be involved in progression of TB disease.
Yeast Models Of Phosphomannomutase 2 Deficiency, A Congenital Disorder Of Glycosylation
Authors: Lao Jessica P. , DiPrimio Nina , Prangley Madeleine , Sam Feba S. , Mast Joshua D. , Perlstein Ethan O.
Published: September 2018

Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeastSaccharomyces cerevisiaegene SEC53. Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations – R141H, F119L respectively– and the most common compound heterozygote genotype – PMM2R141H/F119L– observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.
A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses
Authors: Hausera Felix , Ceciliatoa Paulo H. O. , Lin Yi-Chen , Guo DanDan , Gregerson JD , Abbasi Nazia , Youhanna Diana , Park Jiyoung , Dubeaux Guillaume , Shani Eilon , Poomchongkho Nusra , Schroeder Julian I.
Published: September 2018

The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here we report the generation of over 14,000 amiRNA-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol is developed here for isolating robust and reproducible amiRNA-mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA insensitive amiRNA line is isolated, which targets three genes encoding avirulence-induced gene2-like (AIG2) genes. A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led here to isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 14,000 T2 amiRNA lines generated here provides a new platform for forward genetic screens and is being made available to the Arabidopsis Biological Resource Center (ABRC) and optimized procedures for amiRNA screening and controls are described.

Highlight

The generation of over 14,000 amiRNA-expressing plants is reported that are being made publicly available enabling screens of redundant genes in Arabidopsis. Identification of known and new genes is reported.
A guide to performing Polygenic Risk Score analyses
Authors: Choi Shing Wan , Heng Mak Timothy Shin , O’Reilly Paul F.
Published: September 2018

The application of polygenic risk scores (PRS) has become routine across genetic research. Among a range of applications, PRS are exploited to assess shared aetiology between phenotypes, to evaluate the predictive power of genetic data for use in clinical settings, and as part of experimental studies in which, for example, experiments are performed on individuals, or their biological samples (eg. tissues, cells), at the tails of the PRS distribution and contrasted. As GWAS sample sizes increase and PRS become more powerful, they are set to play a key role in personalised medicine. However, despite the growing application and importance of PRS, there are limited guidelines for performing PRS analyses, which can lead to inconsistency between studies and misinterpretation of results. Here we provide detailed guidelines for performing polygenic risk score analyses relevant to different methods for their calculation, outlining standard quality control steps and offering recommendations for best-practice. We also discuss different methods for the calculation of PRS, common misconceptions regarding the interpretation of results and future challenges.
Foraging as an evidence accumulation process
Authors: Davidson Jacob D. , Hady Ahmed El
Published: September 2018

A canonical foraging task is the patch-leaving problem, in which a forager must decide to leave a current resource in search for another. Theoretical work has derived optimal strategies for when to leave a patch, and experiments have tested for conditions where animals do or do not follow an optimal strategy. Nevertheless, models of patch-leaving decisions do not consider the imperfect and noisy sampling process through which an animal gathers information, and how this process is constrained by neurobiological mechanisms. In this theoretical study, we formulate an evidence accumulation model of patch-leaving decisions where the animal averages over noisy measurements to estimate the state of the current patch and the overall environment. Evidence accumulation models belong to the class of drift diffusion processes and have been used to model decision making in different contexts especially in cognitive and systems neuroscience. We solve the model for conditions where foraging decisions are optimal and equivalent to the marginal value theorem, and perform simulations to analyze deviations from optimal when these conditions are not met. By adjusting the drift rate and decision threshold, the model can represent different “strategies”, for example an increment-decrement or counting strategy. These strategies yield identical decisions in the limiting case but differ in how patch residence times adapt when the foraging environment is uncertain. To account for sub-optimal decisions, we introduce an energy-dependent utility function that predicts longer than optimal patch residence times when food is plentiful. Our model provides a quantitative connection between ecological models of foraging behavior and evidence accumulation models of decision making. Moreover, it provides a theoretical framework for potential experiments which seek to identify neural circuits underlying patch leaving decisions.
SuperCT: A supervised-learning-framework to enhance the characterization of single-cell transcriptomic profiles
Authors: Xie Peng , Gao Mingxuan , Wang Chunming , Noel Pawan , Yang Chaoyong , Hoff Daniel Von , Han Haiyong , Zhang Michael Q. , Lin Wei
Published: September 2018

Characterization of individual cell types is fundamental to the study of multicellular samples such as tumor tissues. Single-cell RNAseq techniques, which allow high-throughput expression profiling of individual cells, have significantly advanced our ability of this task. Currently, most of the scRNA-seq data analyses are commenced with unsupervised clustering of cells followed by visualization of clusters in a low-dimensional space. Clusters are often assigned to different cell types based on canonical markers. However, the efficiency of characterizing the known cell types in this way is low and limited by the investigator[s] knowledge. In this study, we present a technical framework of training the expandable supervised-classifier in order to reveal the single-cell identities based on their RNA expression profiles. Using multiple scRNA-seq datasets we demonstrate the superior accuracy, robustness, compatibility and expandability of this new solution compared to the traditional methods. We use two examples of model upgrade to demonstrate how the projected evolution of the cell-type classifier is realized.
Systematic Prediction of Regulatory Motifs from Human ChIP-Sequencing Data Based on a Deep Learning Framework
Authors: Yang Jinyu , Hoppe Adam D. , Liu Bingqiang , Ma Qin
Published: September 2018

Identification of transcription factor binding sites (TFBSs) and cis-regulatory motifs (motifs for short) from genomics datasets, provides a powerful view of the rules governing the interactions between TFs and DNA. Existing motif prediction methods however, are limited by high false positive rates in TFBSs identification, contributions from non-sequence-specific binding, and complex and indirect binding mechanisms. High throughput next-generation sequencing data provides unprecedented opportunities to overcome these difficulties, as it provides multiple whole-genome scale measurements of TF binding information. Uncovering this information brings new computational and modeling challenges in high-dimensional data mining and heterogeneous data integration. To improve TFBS identification and novel motifs prediction accuracy in the human genome, we developed an advanced computational technique based on deep learning (DL) and high-performance computing, named DESSO. DESSO utilizes deep neural network and binomial distribution to optimize the motif prediction. Our results showed that DESSO outperformed existing tools in predicting distinct motifs from the 690 in vivo ENCODE ChIP-Sequencing (ChIP-Seq) datasets for 161 human TFs in 91 cell lines. We also found that protein-protein interactions (PPIs) are prevalent among human TFs, and a total of 61 potential tethering binding were identified among the 100 TFs in the K562 cell line. To further expand DESSO’s deep-learning capabilities, we included DNA shape features and found that (i) shape information has a strong predictive power for TF-DNA binding specificity; and (ii) it aided in identification of the shape motifs recognized by human TFs which in turn contributed to the interpretation of TF-DNA binding in the absence of sequence recognition. DESSO and the analyses it enabled will continue to improve our understanding of how gene expression is controlled by TFs and the complexities of DNA binding. The source code and the predicted motifs and TFBSs from the 690 ENCODE TF ChIP-Seq datasets are freely available at the DESSO web server: http://bmbl.sdstate.edu/DESSO.
Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform
Authors: Holm Johanna B. , Humphrys Michael S. , Robinson Courtney K. , Settles Matthew L. , Ott Sandra , Fu Li , Yang Hongqiu , Gajer Pawel , He Xin , McComb Elias , Gravitt Patti E , Ghanem Khalil G. , Brotman Rebecca M. , Ravel Jacques
Published: September 2018

Amplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.

Importance

Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.
Two decades of suspect evidence for adaptive DNA-sequence evolution - Failure in consistent detection of positive selection
Authors: He Ziwen , Chen Qipian , Yang Hao , Chen Qingjian , Shi Suhua , Wu Chung-I
Published: September 2018

A recent study suggests that the evidence of adaptive DNA sequence evolution accumulated in the last 20 years may be suspect1. The suspicion thus calls for a re-examination of the reported evidence. The two main lines of evidence are from the McDonald-Kreitman (MK) test, which compares divergence and polymorphism data, and the PAML test, which analyzes multi-species divergence data. Here, we apply these two tests concurrently on the genomic data ofDrosophilaandArabidopsis. To our surprise, the >100 genes identified by the two tests do not overlap beyond random expectations. The results could mean i) high false positives by either test or ii) high false-negatives by both tests due to low powers. To rule out the latter, we merge every 20 - 30 genes into a “supergene”. At the supergene level, the power of detection is high, with 8% - 56% yielding adaptive signals. Nevertheless, the calls still do not overlap. Since it is unlikely that one test is largely correct and the other is mostly wrong (see Discussion), the total evidence of adaptive DNA sequence evolution should be deemed unreliable. As suggested by Chenet al.1, the reported evidence for positive selection may in fact be signals of fluctuating negative selection, which are handled differently by the two tests. Possible paths forward on this central evolutionary issue are discussed.
Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling
Authors: Philip V’kovski , Markus Gerber , Jenna Kelly , Stephanie Pfaender , Nadine Ebert , Sophie Braga Lagache , Cedric Simillion , Jasmine Portmann , Hanspeter Stalder , Véronique Gaschen , Remy Bruggmann , Michael Stoffel , Manfred Heller , Ronald Dijkman , Volker Thiel
Published: September 2018

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses.

Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.
Direct observation of individual tubulin dimers binding to growing microtubules
Authors: Mickolajczyk Keith J. , Geyer Elisabeth A. , Kim Tae , Rice Luke M. , Hancock William O.
Published: September 2018

The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on- and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date.

SIGNIFICANCE

Microtubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on- and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.
neoepiscopeimproves neoepitope prediction with multi-variant phasing
Authors: Wood Mary A. , Nguyen Austin , Struck Adam , Ellrott Kyle , Nellore Abhinav , Thompson Reid F.
Published: September 2018

The vast majority of tools for neoepitope prediction from DNA sequencing of complementary tumor and normal patient samples do not consider germline context or the potential for co-occurrence of two or more somatic variants on the same mRNA transcript. Without consideration of these phenomena, existing approaches are likely to produce both false positive and false negative results, resulting in an inaccurate and incomplete picture of the cancer neoepitope landscape. We developedneoepiscopechiefly to address this issue for single nucleotide variants (SNVs) and insertions/deletions (indels), and herein illustrate how germline and somatic variant phasing affects neoepitope prediction across multiple datasets. We estimate that up to ∼5% of neoepitopes arising from SNVs and indels may require variant phasing for their accurate assessment.neoepiscopeis performant, flexible, and supports several major histocompatibility complex binding affinity prediction tools. We have releasedneoepiscopeas open-source software (MIT license,https://github.com/pdxgx/neoepiscope) for broad use.

KEY POINTS

Germline context and somatic variant phasing are important for neoepitope prediction

Many popular neoepitope prediction tools have issues of performance and reproducibility

We describe and provide performant software for accurate neoepitope prediction from DNA-seq data
Aβ Fibrils Can Act as Aqueous Pores: a Molecular Dynamics Study
Authors: Natesh S. , Sachleben J. R. , Sosnick T. R , Freed K. F. , Meredith S. C. , Haddadian E. J.
Published: September 2018

Aggregation of Aβ peptides is important in the etiology of Alzheimer’s Disease (AD), an increasingly prevalent neurodegenerative disease. We ran multiple ∼ 300 ns all-atom explicit solvent molecular dynamics (MD) simulations starting from three NMR-based structural models of Aβ(1-40 residues) fibrils having 2-fold (pdb code 2LMN) or 3-fold rotational symmetry (2LMP, and 2M4J). The 2M4J structure is based on an AD brain-seeded fibril whereas 2LMP and 2LMN represent two all-synthetic fibrils. Fibrils are constructed to contain either 6 or an infinite number of layers made using periodic images. The 6 layer fibrils partially unravel over the simulation time, mainly at their ends, while infinitely long fibrils do not. Once formed, the D23-K28 salt bridges are very stable and form within and between chains. Fibrils tend to retain (2LMN and 2LMP) or develop (2M4J) a “stagger” or register shift of β-strands along the fibril axis. The brain-seeded fibril rapidly develops gaps at the sides of the fibril, which allows bidirectional flow of water and ions from the bulk phase in and out the central longitudinal core of the fibril. Similar but less marked changes were also observed for the 2LMP fibrils. The residues defining the gaps largely coincide with those demonstrated to have relatively rapid Hydrogen-Deuterium exchange in solid state NMR studies. These observations suggest that Aβ(1-40 residues) fibrils may act as aqueous pores that might disrupt water and ion fluxes if inserted into a cell membrane.
Distributed plasticity drives visual habituation learning in larval zebrafish
Authors: Randlett Owen , Haesemeyer Martin , Forkin Greg , Shoenhard Hannah , Schier Alexander F. , Engert Florian , Granato Michael
Published: September 2018

Habituation is a simple form of learning, where animals learn to reduce their responses to repeated innocuous stimuli. While habituation is simple in concept, its exact implementation in the vertebrate brain is not clear. It could occur via a single plasticity event at a singular site in the circuit, or alternatively via more complex strategies that combine multiple mechanisms at various processing stages and sites. Here, we use a visual habituation assay in larval zebrafish, where larvae habituate to sudden reductions in illumination (dark flashes). We find that 8 different components of this response habituate, including the probability of executing a response, its latency, and measures of its magnitude. Through behavioural analyses, we find that habituation of these different behavioural components occurs independently of each other and at different locations in the circuit. Further, we use genetic and pharmacological manipulations to show that habituation of different behavioural components are molecularly distinct. These results are consistent with a model by which visual habituation originates from the combination of multiple independent processes, which each act to adapt specific components of behaviour. This may allow animals to more specifically habituate behaviour based on stimulus context or internal state.
Cryo-electron microscopy structure of the lipid droplet-formation protein seipin
Authors: Sui Xuewu , Arlt Henning , Brook Kelly P. , Lai Zon Weng , DiMaio Frank , Marks Debora S. , Liao Maofu , Farese Robert V. , Walther Tobias C.
Published: September 2018

Sui et al. report the cryo-EM structure of the conserved luminal domain of the lipid droplet (LD)-formation protein seipin. The structure reveals key features of this domain and suggest a new model for seipin’s role in LD formation.

Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. Here, we report a cryo-electron microscopy structure and functional characterization of D. melanogaster seipin. The structure reveals a ring-shaped dodecamer, with the luminal domain of each monomer resolved at ∼4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix positioned towards the ER bilayer, and a β-sandwich domain that has structural similarity with lipid-binding proteins. This structure, and our functional testing in cells, suggest a model in which seipin oligomers initially detect forming LDs in the ER via hydrophobic helices and subsequently act as membrane anchors to enable lipid transfer and LD growth.
Evaluating FAIR-Compliance Through an Objective, Automated, Community-Governed Framework
Authors: Wilkinson Mark D , Dumontier Michel , Sansone Susanna-Assunta , da Silva Santos Luiz Olavo Bonino , Prieto Mario , McQuilton Peter , Gautier Julian , Murphy Derek , Crosas Mercѐ , Schultes Erik
Published: September 2018

With the increased adoption of the FAIR Principles, a wide range of stakeholders, from scientists to publishers, funding agencies and policy makers, are seeking ways to transparently evaluate resource FAIRness. We describe the FAIR Evaluator, a software infrastructure to register and execute tests of compliance with the recently published FAIR Metrics. The Evaluator enables digital resources to be assessed objectively and transparently. We illustrate its application to three widely used generalist repositories - Dataverse, Dryad, and Zenodo - and report their feedback. Evaluations allow communities to select relevant Metric subsets to deliver FAIRness measurements in diverse and specialized applications. Evaluations are executed in a semi-automated manner through Web Forms filled-in by a user, or through a JSON-based API. A comparison of manual vs automated evaluation reveals that automated evaluations are generally stricter, resulting in lower, though more accurate, FAIRness scores. Finally, we highlight the need for enhanced infrastructure such as standards registries, like FAIRsharing, as well as additional community involvement in domain-specific data infrastructure creation.
RawTools: Rapid and Dynamic Interrogation of Orbitrap Data Files for Mass Spectrometer System Management
Authors: Kovalchik Kevin A. , Colborne Shane , Spencer Sandra , Sorensen P.H. , Chen David D. Y. , Morin Gregg B. , Hughes Christopher S.
Published: September 2018

Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data from Thermo Orbitrap raw files is demonstrated using the RawTools software. RawTools is a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional ‘preview’ database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library, and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.
Different theta frameworks coexist in the hippocampus and are coordinated during memory-guided exploration and novelty detection
Authors: Lopez-Madrona Víctor J. , Pérez-Montoyo Elena , Álvarez-Salvado Efrén , Moratal David , Herreras Oscar , Pereda Ernesto , Mirasso Claudio R. , Canals Santiago
Published: September 2018

Hippocampal firing is organized in theta sequences controlled by internal memory-related processing and by external sensory cues. How these computations are segregated or integrated, depending on the cognitive needs, is not fully understood. Although theta activity in the hippocampus is most commonly studied as a unique coherent oscillation, it is the result of a complex interaction between different rhythm generators. Here we investigated the coordination between theta generators as a possible mechanism to couple or decouple internally and externally driven computations. We separated and quantified three different theta current generators from the hippocampus of freely behaving rats, one originating in CA3 with current sinks in CA1 str. radiatum and two with current sinks in CA1 str. lacunosum-moleculare and dentate molecular layer, mainly driven by entorhinal cortex (EC) layers 3 and 2, respectively. These theta generators followed non fully coherent dynamics and presented epochs of higher and lower phase coupling, suggesting a flexible interaction between them. Selective optogenetic inhibition in CA3 depressed the str. radiatum generator without affecting the EC-driven theta oscillations, indicating that theta rhythm generators can be modulated independently. In addition, band-specific gamma interactions with theta oscillations selectively occurred with the corresponding pathway-specific theta current generator, supporting the existence of different theta-gamma coding frameworks to organize neuronal firing in the hippocampus. Importantly, we found that epochs of highly synchronized theta rhythmicity across generators preferentially occurred during memory-guided exploration and mismatch novelty detection in familiar environments, two conditions in which internally generated memory representations need to be coordinated with the incoming sensory information about external cues. We propose a mechanism for segregating and integrating hippocampal computations based on the coexistence of different theta-gamma frameworks that flexibly couple or decouple accommodating the cognitive needs.