bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and clinical validation of a targeted RNAseq panel (Fusion-STAMP) for diagnostic and predictive gene fusion detection in solid tumors
Authors: Nohr Erik , Kunder Christian A. , Jones Carol , Sutton Shirley , Fung Eula , Zhu Hongbo , Feng Sharon J. , Gojenola Linda , Bustamante Carlos D. , Zehnder James L. , Costa Helio A.
Published: December 2019

RNA sequencing is emerging as a powerful technique to detect a diverse array of fusions in human neoplasia, but few clinically validated assays have been described to date. We designed and validated a hybrid-capture RNAseq assay for FFPE tissue (Fusion-STAMP). It fully targets the transcript isoforms of 43 genes selected for their known impact as actionable targets of existing and emerging anti-cancer therapies (especially in lung adenocarcinomas), prognostic features, and/or utility as diagnostic cancer biomarkers (especially in sarcomas). 57 fusion results across 34 samples were evaluated. Fusion-STAMP demonstrated high overall accuracy with 98% sensitivity and 94% specificity for fusion detection. There was high intra- and inter-run reproducibility. Detection was sensitive to approximately 10% tumor, though this is expected to be impacted by fusion transcript expression levels, hybrid capture efficiency, and RNA quality. Challenges of clinically validating RNA sequencing for fusion detection include a low average RNA quality in FFPE specimens, and variable RNA total content and expression profile per cell. These challenges contribute to highly variable on-target rates, total read pairs, and total mapped read pairs. False positive results may be caused by intergenic splicing, barcode hopping / index hopping, or misalignment. Despite this, Fusion-STAMP demonstrates high overall performance metrics for qualitative fusion detection and is expected to provide clinical utility in identifying actionable fusions.
Rapid and Inexpensive Preparation of Genome-Wide Nucleosome Footprints from Model and Non-Model Organisms
Authors: McKnight Laura E , Crandall Johnathan G , Bailey Thomas B , Banks Orion GB , Orlandi Kona N , Truong Vi N , Waddell Grace L , Wiles Elizabeth T , Donovan Drake A , Hansen Scott D , Selker Eric U , McKnight Jeffrey N
Published: December 2019

Eukaryotic DNA is packaged into nucleosomes, the smallest repeating unit of chromatin. The positions of nucleosomes determine the relative accessibility of genomic DNA. Several protocols exist for mapping nucleosome positions in eukaryotic genomes in order to study the relationship between chromatin structure and DNA-dependent processes. These nucleosome mapping protocols can be laborious and, at minimum, require two to three days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for mapping nucleosomes from S. cerevisiae liquid culture or from patches on solid agar. This method isolates nucleosome-sized footprints in three hours using 1.5 ml tubes with minimal chemical waste. We validate that these footprints match those produced by previously published methods and we demonstrate that our protocol works for N. crassa and S. pombe. A slightly modified protocol can be used for isolation of nucleosome-protected DNA fragments from a variety of wild fungal specimens thereby providing a simple, easily multiplexed and unified strategy to map nucleosome positions in model and non-model fungi. Finally, we demonstrate recovery of nucleosome footprints from the diploid myeloid leukemia cell line PLB-985 in less than three hours using an abbreviated version of the same protocol. With reduced volume and incubation times and a streamlined workflow, the described method should be compatible with high-throughput, automated creation of MNase-seq libraries. We believe this simple validated method for rapidly producing sequencing-ready nucleosome footprints from a variety of organisms will make nucleosome mapping studies widely accessible to researchers globally.
Precise levels of Nectin-3 and an interaction with Afadin are required for proper synapse formation in postnatal visual cortex
Authors: Tomorsky Johanna , Parker Philip R. L. , Doe Chris Q. , Niell Cristopher M.
Published: December 2019

Background

Developing cortical neurons express a tightly choreographed sequence of cytoskeletal and transmembrane proteins to form and strengthen specific synaptic connections during circuit formation. Nectin-3 is a cell-adhesion molecule with previously described roles in synapse formation and maintenance. This protein and its binding partner, Nectin-1, are selectively expressed in upper-layer neurons of mouse visual cortex, but their role in the development of cortical circuits is unknown.

Methods

Here we block Nectin-3 expression (via shRNA) or overexpress Nectin-3 in developing layer 2/3 visual cortical neurons using in utero electroporation. We then assay dendritic spine densities at three developmental time points: eye opening (postnatal day (P)14), one week following eye opening after a period of heightened synaptogenesis (P21), and at the close of the critical period for ocular dominance plasticity (P35).

Results

Knockdown of Nectin-3 beginning at E15.5 or ∼P19 increased dendritic spine densities at P21 or P35, respectively. Conversely, overexpressing full length Nectin-3 at E15.5 led to decreased dendritic spine densities when all ages were considered together. Interestingly, an even greater decrease in dendritic spine densities, particularly at P21, was observed when we overexpressed Nectin-3 lacking its Afadin binding domain, indicating Afadin may facilitate spine morphogenesis after eye opening.

Conclusion

These data collectively suggest that the proper levels of Nectin-3, as well as the interaction of Nectin-3 with Afadin, facilitate normal synapse formation after eye opening in layer 2/3 visual cortical neurons.
VIRAL DELIVERY OF GDNF PROMOTES FUNCTIONAL INTEGRATION OF HUMAN STEM CELL GRAFTS IN PARKINSON’S DISEASE
Authors: Gantner Carlos W , de Luzy Isabelle R , Kauhausen Jessica A , Moriarty Niamh , Niclis Jonathan C , Penna Vanessa , Hunt Cameron P. , Bye Christopher R , Ermine Charlotte M , Pouton Colin W , Kirik Deniz , Thompson Lachlan H , Parish Clare L
Published: December 2019

The derivation of neurotransmitter and region-specific neuronal populations from human pluripotent stem cells (PSC) provides impetus for advancing cell therapies into the clinic. At the forefront is our ability to generate ventral midbrain (VM) dopaminergic (DA) progenitors, suitable for transplantation in Parkinson’s disease (PD). Pre-clinical studies, however, have highlighted the low proportion of DA neurons within these grafts and their inferior plasticity by comparison to human fetal donor transplants. Here we sought to examine whether modification of the host environment, through viral delivery of a developmentally critical molecule, glial cell line-derived neurotrophic factor (GDNF), could improve graft survival, integration and function in Parkinsonian rodents. Utilising LMX1A- and PITX3-GFP hPSC reporter lines, we tracked the response of DA progenitors implanted into either a GDNF-rich environment, or in a second group, after a 3-week delay in onset of exposure. We found that early exposure of the graft to GDNF promoted survival of DA and non-DA cells, leading to enhanced motor recovery in PD rats. Delayed overexpression of intrastriatal GDNF also promoted motor recovery in transplanted rats, through alternate selective mechanisms including enhanced A9/A10 specification, increased DA graft plasticity, greater activation of striatal neurons and elevated DA metabolism. Lastly, transcriptional profiling of the grafts highlighted novel genes underpinning these changes. Collectively these results demonstrate the potential of targeted neurotrophic gene therapy strategies to improve human PSC graft outcomes.
Widespread interaction between ADAR1 and transcriptional byproducts
Authors: Wu Chan-Shuo , Tang Sze Jing , Tan Hong Kee , Hung Li-Yuan , Teo Wei Wen , Li Jia , An Omer , Tan Kar Tong , Zhou Qiling , Chen Leilei , Tenen Daniel G. , Yang Henry
Published: December 2019

Background

ADAR1, an adenosine-to-inosine (A-to-I) RNA editing enzyme, has an emerging role in cancer immunotherapy. ADAR1 presumably works by suppressing cellular innate immunity response to endogenously generated double-stranded RNAs through RNA editing. However, RNA species that are directly regulated by ADAR1 mediated RNA editing processes remain poorly defined.

Results

In this study, we used a novel bioinformatics approach to track ADAR1-RNA interactions. By integrating DNA-seq, RNA-seq, and ADAR1 RNA immunoprecipitation sequencing (fRIP-seq) data of K562 cell line, we provided the first in-situ landscape profiling of ADAR1 RNA binding and editing activities. With long RNA fragments captured by ADAR1 immunoprecipitation, we were able to identify exon junctions and genomic boundaries used by ADAR1-associated RNAs and thus we could possibly trace pre-RNA processing steps that had been acting on them. Our methodology allowed us to acquire the knowledge of transcriptome-wide scenario of ADAR1 activities. Intriguingly, we found that ADAR1 had a tendency to interact with transcriptional byproducts originated from obscure regions such as introns and intergenic regions.

Conclusions

Our observation might shed light on the dual role of ADAR1 proteins not only in diversifying the transcriptome, but also in reigning RNA debris from obscure regions. Moreover, as the functional potential of seemly transcriptional byproducts is just beginning to emerge, this study would bridge ADAR1 with other fields of RNA biology.
Microbial residents of the Atlantis Massif’s shallow serpentinite subsurface
Authors: Motamedi Shahrzad , Orcutt Beth N. , Früh-Green Gretchen L. , Twing Katrina I. , Pendleton H. Lizethe , Brazelton William J.
Published: December 2019

The Atlantis Massif rises 4,000 m above the seafloor near the Mid-Atlantic Ridge and consists of rocks uplifted from Earth’s upper mantle. Exposure of the mantle rocks to seawater leads to their alteration into serpentinites. These aqueous geochemical reactions, collectively known as the process of serpentinization, are exothermic and are associated with the release of hydrogen gas (H2), methane (CH4), and small organic molecules. The biological consequences of this flux of energy and organic compounds from the Atlantis Massif were explored by International Ocean Discovery Program (IODP) Expedition 357, which used seabed drills to collect continuous sequences of shallow (<16 meters below seafloor) marine serpentinites and mafic assemblages. Here, we report the first census of microbial diversity in samples of the drill cores, as measured by environmental 16S rRNA gene amplicon sequencing. The problem of contamination of subsurface samples was a primary concern during all stages of this project, starting from the initial study design, continuing to the collection of samples from the seafloor, handling the samples shipboard and in the lab, preparing the samples for DNA extraction, and analyzing the DNA sequence data. To distinguish endemic microbial taxa of serpentinite subsurface rocks from seawater residents and other potential contaminants, the distributions of individual 16S rRNA gene sequences among all samples were evaluated, taking into consideration both presence/absence as well as relative abundances. Our results highlight a few candidate residents of the shallow serpentinite subsurface, including uncultured representatives of the Thermoplasmata, Acidobacteria, Acidimicrobiia, and Chloroflexi.

Importance

International Ocean Discovery Program Expedition 357: “Serpentinization and Life” utilized seabed drills for the first time to collect rocks from the oceanic crust. The recovered rock cores represent the shallow serpentinite subsurface of the Atlantis Massif, where reactions between uplifted mantle rocks and water, collectively known as serpentinization, produce environmental conditions that can stimulate biological activity and are thought to be analogous to environments that were prevalent on the early Earth and perhaps other planets. The methodology and results of this project have implications for life detection experiments, including sample return missions, and provide the first window into the diversity of microbial communities inhabiting subseafloor serpentinites.
Na2CO3-Responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics
Authors: Suo Jinwei , Zhang Heng , Zhao Qi , Zhang Nan , Zhang Yongxue , Li Ying , Song Baohua , Yu Juanjuan , Cao Jianguo , Wang Tai , Luo Ji , Guo Lihai , Ma Jun , Zhang Xumin , She Yimin , Peng Lianwei , Ma Weimin , Guo Siyi , Miao Yuchen , Chen Sixue , Qin Zhi , Dai Shaojun
Published: December 2019

Alkali-salinity exerts severe osmotic, ionic and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, and they have improved our understanding of the Na2CO3-responsive mechanisms in halophytes.
Time and mode of Culicidae evolutionary history
Authors: da Silva Alexandre Freitas , Machado Laís Ceschini , de Paula Marcia Bicudo , Vieira Carla Júlia da Silva Pessoa , Bronzoni Roberta Vieira de Morais , de Melo Santos Maria Alice Varjal , Wallau Gabriel Luz
Published: December 2019

Mosquitoes are insects of medical importance due their role as vectors of different pathogens to humans and other mammals. There is a lack of information about the evolutionary history and phylogenetic positioning of the majority of mosquitoes species. Here we sequenced the mitogenomes of mosquitoes species through low-coverage sequencing and data mining. A total of 37 draft mitogenomes were assembled representing 11 genera and 16 of those were sequenced for the first time. The recovered mitogenomes showed a coverage breadth average of 81.24%. Most of the species were clustered in monophyletic clades with other members of their own genus with exception of the Aedini tribe which was paraphyletic corroborating other findings. We established for the first time the monophyletic status of eight species from the tribe Mansoniini including both Coquillettidia and Mansonia genus and established the basal positioning of Aedeomyiini and Uranotaeniini tribes regarding the Culicinae subfamily. Molecular clock dated the Culicidae family emergence around 273 MYA and the split between Anophelinae and Culicinae subfamily around 182 MYA in the Jurassic period. Low-coverage sequencing is effective to recover mitogenomes, establish phylogenetic knowledge generating basic fundamental information to the understanding of the role of these species as pathogen vectors.
Genetic risk of dementia modifies the impact of obesity on limbic white matter and spatial navigation behavior in cognitively healthy adults
Authors: Mole Jilu P. , Fasano Fabrizio , Evans John , Sims Rebecca , Hamilton Derek A. , Kidd Emma , Metzler-Baddeley Claudia
Published: December 2019

A family history (FH) of dementia,APOE-ε4 genotype, and obesity are major risk factors for developing Alzheimer’s disease but their combined effects on the brain and cognition remain elusive. We tested the hypothesis that these risk factors affect apparent white matter (WM) myelin and cognition including spatial navigation and processing speed in 166 asymptomatic individuals (38-71 years). Microstructure in temporal [fornix, parahippocampal cingulum, uncinate fasciculus], motor and whole-brain WM was assessed with myelin-sensitive indices from quantitative magnetization transfer [macromolecular proton fraction (MPF)] and axon density from diffusion imaging. Individuals with the highest genetic risk (FH+ andAPOE-ε4) compared to those with FH+ alone showed obesity-related reductions in MPF and axon density in the right parahippocampal cingulum. No effects were present for those without FH. Furthermore, FH modulated obesity-related effects on spatial navigation behaviour. In summary, an individual’s genetic dementia risk influenced the impact of obesity on WM myelin and cognition.
Genomic and transcriptomic evidence for descent from Plasmodium and loss of blood schizogony in Hepatocystis parasites from naturally infected red colobus monkeys
Authors: Aunin Eerik , Böhme Ulrike , Sanderson Theo , Simons Noah D , Goldberg Tony L , Ting Nelson , Chapman Colin A , Newbold Chris I , Berriman Matthew , Reid Adam J
Published: December 2019

Hepatocystis is a genus of single-celled parasites infecting monkeys, bats and squirrels. Although thought to descend from malaria parasites (Plasmodium spp.), Hepatocystis spp. are thought not to undergo replication in the blood – the part of the Plasmodium life cycle which causes the symptoms of malaria. Furthermore, Hepatocystis is transmitted by midges, not mosquitoes. Comparative genomics of Hepatocystis and Plasmodium species therefore presents an opportunity to better understand some of the most important aspects of malaria parasite biology. We were able to generate a draft genome for Hepatocystis using DNA sequencing reads from the blood of a naturally infected red colobus monkey. We provide robust phylogenetic support for Hepatocystis as a sister group to Plasmodium parasites infecting rodents. We show transcriptomic support for a lack of replication in the blood and genomic support for a complete loss of a family of genes involved in red blood cell invasion. Our analyses highlight the rapid evolution of genes involved in parasite vector stages, revealing genes that may be critical for interactions between malaria parasites and mosquitoes.
Overexpression of Mig-6 in Limb Mesenchyme Leads to Accelerated Osteoarthritis in Mice
Authors: Bellini Melina Rodrigues , Pest Michael Andrew , Jeong Jae-Wook , Beier Frank
Published: December 2019

Background

Mitogen-inducible gene 6 (Mig-6) is a tumour suppressor gene that is also associated with the development of osteoarthritis (OA)-like disorder. Recent evidence from our lab and others showed that cartilage-specific Mig-6 knockout (KO) mice develop chondro-osseous nodules, along with increased articular cartilage thickness and enhanced EGFR signaling in the articular cartilage. Here, we evaluate the phenotype of mice with skeletal-specific overexpression of Mig-6.

Methods

Synovial joint tissues of the knee were assessed in 12 and 36 weeks-old skeleton-specificMig-6overexpressing (Mig-6over/over) and control animals using histological stains, immunohistochemistry, semi-quantitative OARSI scoring, and microCT for skeletal morphometry. Measurement of articular cartilage and subchondral bone thickness were also performed using histomorphometry.

Results

Our results show only subtle developmental effects of Mig-6 overexpression. However, maleMig-6over/overmice show accelerated cartilage degeneration at 36 weeks of age, in both medial and lateral compartments of the knee. Immunohistochemistry for SOX9 and PRG4 showed decreased staining inMig-6over/overmice relative to controls, providing potential molecular mechanisms for the observed effects.

Conclusion

Overexpression ofMig-6in articular cartilage causes no major developmental phenotype but results in accelerated development of OA during aging. These data demonstrate that precise regulation of the Mig-6/EGFR pathway is critical for joint homeostasis.
Optimizing an eDNA protocol for monitoring endangered Chinook Salmon in the San Francisco Estuary: balancing sensitivity, cost and time
Authors: Sanches Thiago M. , Schreier Andrea M.
Published: December 2019

Environmental DNA (eDNA) analysis has gained traction as a precise and cost effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for a given environmental condition.

Author Summary

The use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent years. Though, due to differences in the ecology of the environment studied and the novelty of the technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide a concise interpretation of best practices given different project constraints. For the conditions of the San Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support for managers to decide their standards ahead of project submission not only for estuarine conditions but for other waterine conditions alike.
Laboratory testing of very low-copper-treated water to prolong pupation and emerging time of mosquito larvae: an alternative method to delay mosquito breeding capability
Authors: Reza Mohamad , Ilmiawati Cimi
Published: December 2019

Introduction

Larvicide application in ovitrap is one of the currently available methods used in mosquito eradication campaign because they eliminate the larval stage. We previously reported that copper in liquid form is a promising candidate due to its potent larvicide properties in a laboratory setting and even in the field. In the field study, several larvae survived in the ovitrap due to the dilution of copper concentration by the rain. The surviving larvae were smaller and less motile. This led our interest to study the effect of a sub-lethal dose of copper in ovitrap on larvae development, pupation time and lifespan in the adult stage.

Methods

First instar larvae of three species of mosquito (Aedes albopictus, Culex pipiens and Anopheles stephensi) were bred in copper-treated water at a concentration of 0.60 ppm, 0.30 ppm, and 0.15 ppm and compared with the control group. The surviving larvae were recorded every day in terms of pupa emerging time and adult emerging time. The number of adult mortality was recorded and compared with the control.

Results

Copper showed potent larvicide effect in the 0.60 ppm concentration and prolonged pupation time and caused a significantly lower number of emerging mosquitoes down to the lowest concentration of 0.15 ppm. The adult lifespan was not different compared to the control.

Conclusion

This study demonstrates the capability of copper below 1 ppm to prolong the pupation time and the emerging time of mosquito larvae. Our findings open the possibility of copper application to cut mosquito breeding capacity that eventually will reduce disease transmission.
Developing political-ecological theory: The need for Many-Task Computing
Authors: Haas Timothy C.
Published: December 2019

Models of political-ecological systems can inform policies for managing ecosystems that contain endangered species. One way to increase the credibility of these models is to subject them to a rigorous suite of data-based statistical assessments. Doing so involves statistically estimating the model’s parameters, computing confidence intervals for these parameters, determining the model’s prediction error rate, and assessing its sensitivity to parameter misspecification.

Here, these statistical algorithms along with a method for constructing politically feasible policies from a statistically fitted model, are coded as JavaSpaces™ programs that run as compute jobs on either supercomputers or a collection of in-house workstations. Several new algorithms for implementing such jobs in distributed computing environments are described.

This downloadable code is used to compute each job’s output for the management challenge of conserving the East African cheetah (Acinonyx jubatus). This case study shows that the proposed suite of statistical tools can be run on a supercomputer to establish the credibility of a managerially-relevant model of a political-ecological system that contains one or more endangered species. This demonstration means that the new standard of credibility that any political-ecological model needs to meet before being used to inform ecosystem management decisions, is the one given herein.
Comparative proximity biotinylation implicates RAB18 in sterol mobilization and biosynthesis
Authors: Kiss Robert S. , Chicoine Jarred , Khalil Youssef , Sladek Robert , Chen He , Pisaturo Alessandro , Martin Cyril , Dale Jessica D. , Brudenell Tegan A. , Kamath Archith , Paci Emanuele , Kerksiek Anja , Lütjohann Dieter , Clayton Peter , Wills Jimi C. , von Kriegsheim Alex , Nilsson Tommy , Sheridan Eamonn , Handley Mark T.
Published: December 2019

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18-interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. 12 of these 28 interactions are supported by prior reports and we have directly validated novel interactions with SEC22A, TMCO4 and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP (emopamil binding protein) is a Δ8-Δ7 sterol isomerase and OSBPL2/ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We find that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Further,de novocholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.
Effective buprenorphine use and tapering strategies: Endorsements and insights by people in recovery from opioid use disorder on a Reddit forum
Authors: Graves Rachel L , Sarker Abeed , Al-Garadi Mohammed Ali , Yang Yuan-chi , Love Jennifer S , O’Connor Karen , Gonzalez-Hernandez Graciela , Perrone Jeanmarie
Published: December 2019

Opioid use disorder (OUD) is a public health emergency in the United States. Over 47,000 overdose-related deaths in 2017 involved opioids. Medication-assisted treatment (MAT), in particular, buprenorphine and buprenorphine combination products such as Suboxone®, is the most effective, evidence-based treatment for OUD. However, there are a limited number of conclusive scientific studies that provide guidance to medical professionals about strategies for using buprenorphine to achieve stable recovery. In this study, we used data-driven natural language processing methods to mine a total of 16,146 posts about buprenorphine from 1933 unique users on the anonymous social network Reddit. Analysis of a sample of these posts showed that 74% of the posts described users’ personal experiences and that the top three topics included advice on using Suboxone® (55.0%), Suboxone® dosage information (35.5%) and information about Suboxone® tapering (32.0%). Based on two models, one that incorporated ‘upvoting’ by other members and one that did not, we found that Reddit users reported more successful recovery with longer tapering schedules, particularly from 2.0 mg to 0.0 mg (median: 93 days; mean: 95 days), as compared to shorter tapering schedules investigated in past clinical trials. Diarrhea, insomnia, restlessness, and fatigue were commonly reported adverse events. Physical exercise, clonidine, and Imodium® were frequently reported to help during the recovery process. Due to the difficulties of conducting longer-term clinical trials involving patients with OUD, clinicians should consider other information sources including peer discussions from the abundant, real-time information available on Reddit.

Significance Statement

Opioid use disorder (OUD) is a national crisis in the United States and buprenorphine is one of the most effective evidence-based treatments. However, few studies have explored successful strategies for using and tapering buprenorphine to achieve stable recovery, particularly due to the difficulties of conducting long-term studies involving patients with OUD. In this study, we show that discussions on the anonymous social network Reddit may be leveraged, via automatic text mining methods, to discover successful buprenorphine use and tapering strategies. We discovered that longer tapering schedules, compared to those investigated in past clinical trials, may lead to (self-reported) sustained recovery. Furthermore, Reddit posts also provide key information regarding buprenorphine withdrawal, cravings, adjunct medications for withdrawal symptoms and relapse prevention strategies.
eVIP2: Expression-based variant impact phenotyping to predict the function of gene variants
Authors: Thornton Alexis M. , Fang Lishan , O’Brien Casey , Berger Alice H. , Giannakis Marios , Brooks Angela N.
Published: December 2019

While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659, was, surprisingly, predicted to be a gain-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF alpha via NFKB signaling, KRAS signaling, and hypoxia. To validate these predictions, we performed reporter assays and found that all eVIP2 impactful pathways tested in the assay were activated by expression of RNF43 G659fs, but not by expression of RNF43 WT, supporting that RNF43 G659fs is a gain-of-function mutation and its effect on the identified pathways. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.
A Higher Proportion of Craniosynostosis Genes Are Cancer Driver Genes
Authors: Misra Suchir , Shih Andrew , Yan Xiao-Jie , Li Wentian
Published: December 2019

Craniosynostosis (CRS) is a congenital abnormality deformity with a heterogenous genetic contribution. Previously, there are two attempts to collect genes that are genetically associated with craniosynostosis and some related syndromes with 57 (Twigg and Wilkie, 2015) and 39 (Goos and Mathijssen, 2019) genes identified, respectively. We expanded this list of craniosynostosis genes by adding another 17 genes with an updated literature search. These genes are shown to be more likely to be intolerant to functional mutations. Of these 113 craniosynostosis genes, 21 (19% vs. 1.5% baseline frequency) are cancer driver genes, a 14-fold enrichment. The cancer-craniosynostosis connection is further validated by an over-representation analysis of craniosynostosis genes in KEGG cancer pathway and several cancer related gene-sets. Many cancer-craniosynostosis overlapping genes participate in intracellular signaling pathways, which play a role in both development and cancer. This connection can be viewed from the oncogenesis recapitulates ontogenesis framework. Nineteen craniosynostosis genes are transcription factor genes (16.8% vs. 8.2% baseline), and craniosynostosis genes are also enriched in targets of certain transcription factors or micro RNAs.
Complex deletion and proximal reinsertion of a 150bp regulatory sequence in the mouse Csf1r promoter mediated by CRISPR-Cas9
Authors: Summers Kim M. , Pridans Clare , Wollscheid-Lengeling Evi , Grabert Kathleen , Adamson Antony , Humphreys Neil E. , Irvine Katharine M. , Hume David A.
Published: December 2019

This paper describes a deletion/reinsertion event encountered in a genome-editing project using CRISPR-Cas9. The objective was to delete a 150bp enhancer region in the mouse Csf1r locus using a pair of guides and a homology-dependent repair (HDR) template. The editing was successful in generating a founder pup with the anticipated precise deletion. However, the deleted fragment and a duplicated copy of part of the HDR template was reinserted around 50bp downstream. The reinsertion event was recognised because the PCR primer site used in genotyping was duplicated, so that there were three PCR products in a heterozygous animal and two in a homozygote. The event we describe is more subtle and more difficult to detect than large-scale rearrangements reported by others. We suggest that any genomic deletion mediated by CRISPR-Cas9 needs to be confirmed by assessing the copy number in the genome.
The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface
Authors: Oliveira Paiva Ana M. , de Jong Leen , Friggen Annemieke H. , Smits Wiep Klaas , Corver Jeroen
Published: December 2019

Clostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold.

In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors.

Importance

Transcription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.

Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain.

Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.
The Min system disassembles FtsZ foci and inhibits polar peptidoglycan remodeling inBacillus subtilis
Authors: Yu Yuanchen , Zhou Jinsheng , Dempwollf Felix , Baker Joshua D. , Kearns Daniel B. , Jacobson Stephen C.
Published: December 2019

A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ inBacillus subtilis. Consistent with previous reports, we find that after division FtsZ rings remain at the cell pole, and FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated forminD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in theminDmutant results in maintenance of multiple Z-rings simultaneously, that are restricted by competition for newly synthesized FtsZ. Whereas the parameters of FtsZ dynamics change in theminDmutant, the overall cell cycle remains the same, albeit with elongated cells necessary to accumulate a threshold concentration of FtsZ for promoting medial division. Finally, theminDmutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in theminDmutant suggesting that not only is the Min system important for recycling FtsZ but also may have a secondary role in the regulation of peptidoglycan remodeling.

IMPORTANCE

Many bacteria grow and divide by binary fission in which a mothercell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyze FtsZ dynamics during growth and find that the Min system ofBacillus subtilisis essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the “inert” property of cell walls at the poles.
Structure of the processive human Pol δ holoenzyme
Authors: Lancey Claudia , Tehseen Muhammad , Raducanu Vlad-Stefan , Rashid Fahad , Merino Nekane , Ragan Timothy J. , Savva Christos , Zaher Manal S. , Shirbini Afnan , Blanco Francisco J. , Hamdan Samir M. , Biasio Alfredo De
Published: December 2019

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ’s activity in replicating the human genome.
Rebar corrosion behavior of coral aggregate seawater concrete by electrochemical techniques
Authors: Wu Zhangyu , Yu Hongfa , Ma Haiyan , Da Bo , Tan Yongshan
Published: December 2019

Purpose

Coral aggregate seawater concrete (CASC) is a new type of lightweight aggregate concrete that is becoming widely used in reef engineering. To investigate the corrosion behavior of different kinds of rebar in CASC exposed to simulated seawater for 0-270 d, the electrochemical techniques, including linear polarization resistance (LPR) technique and the electrochemical impedance spectroscopy (EIS), were used in the present work.

Design/methodology/approach

The electrochemical techniques, including LPR technique and the EIS, were used in the present work.

Findings

Based on the time-varying law of linear polarization curves, self-corrosion potential (Ecorr), polarization resistance (Rp), corrosion current density (Icorr), corrosion rate (i), and the characteristics of EIS diagrams for different types of rebar in CASC, it can be found that the anti-corrosion property of them can be ranked as epoxy resin coated steel > 2205 duplex stainless steel (2205S) > 316 L stainless steel (316 L) > organic coated steel > ordinary steel. Additionally, the linear regression equation between Rp and charge transfer resistance (Rct) was established. Finally, the EIS corrosion standard of rebar was established from the LPR corrosion standard, which provides a direct standard for the EIS technique to determine the condition of rebar in CASC.

Originality/value

The linear regression equation between polarization resistance and charge transfer resistance was established. And the EIS corrosion standard of rebar was established from the LPR corrosion standard, which provides a direct standard for the EIS technique to determine the condition of rebar in CASC.
Antecedents of work readjustment of professional returnees: evidence from Vietnam
Authors: Hoang Hung Trong , Ho Nga Thi Thuy
Published: December 2019

Purpose

The purpose of this paper is to investigate the factors influencing work readjustment of Vietnamese returnees who used to study and/or work in a developed country and are currently working in different positions in their home country.

Design/methodology/approach

Data for this study were collected through a survey of 433 returnees using both paper-based and online surveys. Multiple regression was used to test the relationships in the model.

Findings

The findings show that while the length of time spent overseas, work expectations and subjective norm significantly affect work readjustment, the influences of age, gender and length of time since return on work readjustment are not supported.

Practical implications

The findings provide useful insights for home country government and managers of returnees developing repatriation programs that help returnees deal with the issue of poor work readjustment.

Originality/value

Empirical studies on cross-cultural re-entry adjustment of both self-initiated repatriates and international students are scarcely investigated. Most prior studies focused on individual factors (such as gender, age, duration in overseas and since return), research on the effect of work expectation on work readjustment is still scant. Most prior studies focused on examining the relationship between work expectation and work readjustment of company repatriates, however, this relationship in the context of returnees, especially in the Asia-Pacific region, has not been investigated. Furthermore, this study is the first to examine the influence of subjective norm on work readjustment of returnees.
The degree of the uncertain reward and customer engagement
Authors: Guo Zhaoyang , Zhang Yuan , Zhang Yirang , Ke Xue
Published: December 2019

Purpose

As value co-creation has become a critical strategy for companies to gain competitive advantage, the purpose of this paper is to investigate the degree of the uncertain reward (DUR) and its impact on customer engagement (CE), particularly in the promotion stage, which has not been sufficiently explored. Further, optimistic estimation (OE) is examined as an underlying mechanism of the uncertain reward effect, as well as the impact of combining this with other marketing strategies: the controllable lottery and the delayed reward.

Design/methodology/approach

Three studies were conducted to examine the influence of DUR on CE, which included online experiments and a laboratory experiment. In total, 337 participants were recruited from China and the USA to enhance the study’s reliability and validity.

Findings

The research demonstrated that a high-degree uncertain reward led to less OE than a low-degree uncertain reward (LDUR), which subsequently decreased CE (Study 1). However, when other marketing strategies were combined – the controllable lottery (Study 2) and time-delay reward strategy (Study 3) – the uncertain reward effect was reversed (Study 2) or attenuated (Study 3).

Research limitations/implications

The current research only presents two possible reward amounts and independently explores the influence of two popular marketing strategies. Future research can explore customers’ responses to engagement when they face multiple rewards and thoroughly investigate the influence of other social or psychological factors.

Practical implications

Firms could apply an LDUR to enhance CE effects. Furthermore, this could be done at a low cost by empowering customers’ controllability. Nonetheless, firms should be cautious with trade-offs when using time-delay reward strategies.

Social implications

The research contributes to establishing networks of customer–company and interpersonal relationships, as well as fostering closer social ties and social harmony.

Originality/value

This research offers not only initial research on CE in the promotion stage, but also a novel psychological perspective in CE literature. Meanwhile, the study provides substantial value in guiding managers to effectively transform customers into value co-creators.