bluuebunny/crossref_metadata_2025_split huggingface dataset:
Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish
Authors: Prykhozhij Sergey V. , Fuller Charlotte , Steele Shelby L. , Veinotte Chansey J. , Razaghi Babak , Robitaille Johane , McMaster Christopher , Shlien Adam , Malkin David , Berman Jason N. 
Published: September 2017

AbstractSingle-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations intp53,cdh5andlmnaas case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of twotp53knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for bothtp53knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency fortp53mutants was likely due to the distance between the Cas9 cut site and mutations sincecdh5G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.
Traditional NorwegianKveikYeasts: Underexplored DomesticatedSaccharomyces cerevisiaeYeasts
Authors: Preiss Richard , Tyrawa Caroline , van der Merwe George 
Published: September 2017

AbstractHuman activity has resulted in the domestication ofSaccharomyces cerevisiaeyeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a home-brewing culture in western Norway which has passed down yeasts referred to askveikfor generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavour negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally,kveikyeasts are highly temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>30 °C) wort. Here, we characterizekveikyeasts from 9 different Norwegian sources via PCR fingerprinting, phenotypic screens, lab-scale fermentations, and flavour metabolite analysis using HS-SPME-GC-MS. Genetic fingerprinting via interdelta PCR suggests thatkveikyeasts form a lineage distinct from other domesticated yeasts. Our analyses confirm thatkveikyeasts display hallmarks of domestication such as loss of 4-vinylguaiacol production and high flocculation, and show superior thermotolerance, ethanol tolerance, fermentation rate, and unique flavour metabolite production profiles in comparison to other ale strains, suggesting a broad industrial potential for this group of yeasts.
Cdt1 modulates kinetochore-microtubule attachment stabilization via an Aurora B kinase-dependent mechanism
Authors: Agarwal Shivangi , Smith Kyle Paul , Zhou Yizhuo , Suzuki Aussie , McKenney Richard J. , Varma Dileep 
Published: September 2017

AbstractRobust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner, leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region was required for efficient MT-binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT-binding and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.eTOC summary• Cdt1 binds to microtubules• The middle and the C-terminal winged-helix domains of Cdt1 are involved in MT-binding• Aurora B Kinase phosphorylates Cdt1 and influences its MT-binding• Aurora B-mediated Cdt1 phosphorylation is necessary for kMT stability and mitotic progression
Entering the era of conservation genomics: Cost-effective assembly of the African wild dog genome using linked long reads
Authors: Armstrong Ellie E. , Taylor Ryan W. , Prost Stefan , Blinston Peter , der Meer Esther van , Madzikanda Hillary , Mufute Olivia , Mandisodza Roseline , Stuelpnagel John , Sillero-Zubiri Claudio , Petrov Dmitri 
Published: September 2017

AbstractA high-quality reference genome assembly is a valuable tool for the study of non- model organisms across disciplines. Genomic techniques can provide important insights about past population sizes, local adaptation, and even aid in the development of breeding management plans. This information can be particularly important for fields like conservation genetics, where endangered species require critical and immediate attention. However, funding for genomic-based methods can be sparse for conservation projects, as costs for general species management can consume budgets. Here we report the generation of high-quality reference genomes for the African wild dog (Lycaon pictus) at a low cost, thereby facilitating future studies of this endangered canid. We generated assemblies for three individuals from whole blood samples using the linked-read 10x Genomics Chromium system. The most continuous assembly had a scaffold N50 of 21 Mb, a contig N50 of 83 Kb, and completely reconstructed 95% of conserved mammalian genes as reported by BUSCO v2, indicating a high assembly quality. Thus, we show that 10x Genomics Chromium data can be used to effectively generate high-quality genomes of mammal species from Illumina short-read data of intermediate coverage (∼25-50x). Interestingly, the African wild dog shows a much higher heterozygosity than other species of conservation concern, possibly as a result of its behavioral ecology. The availability of reference genomes for non-model organisms will facilitate better genetic monitoring of threatened species such as the African wild dog. At the same time, they can help researchers and conservationists to better understand the ecology and adaptability of those species in a changing environment.
Selection of DNA Aptamer That Blocks the Fibrillogenesis of a Proteolytic Amyloidogenic Fragment of β2m
Authors: Fukasawa Kanon , Higashimoto Yuichiro , Ando Yukio , Motomiya Yoshihiro 
Published: September 2017

AbstractDialysis‐related amyloidosis (DRA) is a severe complication of hemodialysis that results in progressive destruction of bones and joints. Elevated concentrations of the β2‐microglobulin (β2m) level in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in osteoarticular tissues. β2m lacking the N‐terminal six residues of the mature protein (ΔN6β2m) constitutes 25–30% of β2m in ex vivo DRA amyloid. Unlike full‐length wild‐type β2m, ΔN6β2m forms amyloid fibrils at neutral pH in vitro. However, the role of ΔN6β2m in DRA is, at present, poorly understood. In the present study, we screened novel phosphorothioate‐modified aptamers directed against ΔN6β2m using combinatorial chemistry in vitro. We identified 11 ΔN6β2m aptamers; among the identified aptamers, clone #2, #8, and #10 aptamers had higher binding affinity to ΔN6β2m than the others. Biolayer interferometry analysis revealed that KD values of clone #2, #8, and #10 aptamers were 56, 23, and 44 nM, respectively. Furthermore, the clone #8 aptamer inhibited fibril formation in a dose‐dependent manner, as assessed by Thioflavin T fluorescence assay. Fibrils formed from ΔN6β2m bind to Congo red, displaying changes in the absorbance spectrum of the dye characteristic of binding to amyloid fibrils, which was completely blocked by treatment with clone #8 aptamer. These results suggest the potential of ΔN6β2m aptamers as tools for elucidating co‐assembly mechanisms in amyloid formation.
Endoscopic sleeve gastroplasty and its application to China
Authors: Badurdeen Dilhana S , Kumbhari Vivek 
Published: September 2017

Obesity is a burgeoning problem in China with potential for significant adverse economic and health consequences if not addressed. Unfortunately, the body mass index (BMI) targets used in the West are not applicable to China as Asians have visceral adiposity and thus develop complications of obesity at a lower BMI. Recent studies suggest using lower BMI targets for defining overweight and obesity and to qualify patients for bariatric surgery. Diet, exercise and pharmacology have been the foundation for treatment in those not meeting criteria for bariatric surgery, despite modest weight loss that is poorly sustained over time.The large majority in China are averse to bariatric surgery and thus endoscopic bariatric therapy is emerging as an illustrious remedy to fill this gap. Of the current procedures available endoscopic sleeve gastroplasty has shown the most promise due to the significant and sustained weight loss it produces coupled with the ease of execution and lower cost.The procedure is performed under general anesthesia using a full‐thickness endoscopic suturing device and patients are discharged the same day. The mean procedure time in experienced hands is 60 minutes. Due to the repetitive nature of the procedure, the learning curve is steep. Data thus far demonstrate total body weight loss to be 16–20% at 12 months. The safety profile is excellent with a low morbidity rate seen in the 3 000 procedures performed worldwide. Therefore, widespread dissemination in China is expected with the potential to help millions of obese patients.
Does previous caesarean section at full dilatation increase the likelihood of subsequent spontaneous preterm birth?
Authors: Cong Angela , de Vries Bradley , Ludlow Joanne 
Published: September 2017

BackgroundThere is emerging evidence that caesarean section at full dilatation is associated with an increased risk of subsequent spontaneous preterm birth.AimTo investigate the association between caesarean section at full dilatation and spontaneous preterm birth in subsequent pregnancies.Materials and methodsThis was a retrospective cohort study of women who had two consecutive births at Royal Prince Alfred Hospital, 1989–2015. Our main comparison group was women who had emergency caesarean sections during the first stage of labour. Secondary comparison groups were women who had elective caesarean sections, instrumental deliveries and unassisted vaginal deliveries. The primary outcome was spontaneous preterm birth (<37 weeks gestation) in a subsequent pregnancy.ResultsThere were 2672 women who had an emergency caesarean section, with 2142 (80%) performed during the first stage of labour and 533 (20%) at full dilatation. The rates of spontaneous preterm birth in a subsequent pregnancy were 1.7% and 3.8%, respectively (odds ratio 2.2 (95%CI 1.3–3.8), P = 0.003). The hazard ratio for spontaneous onset of labour at any given gestation from 20 weeks until full term was 1.4 (95%CI 1.2–1.6) and did not change after adjusting for maternal age and body mass index.ConclusionThere is a significantly higher rate of subsequent spontaneous preterm birth in women who had a caesarean section at full dilatation compared with women who had a caesarean section during the first stage of labour. Awareness of this as a risk factor may warrant referral to a high‐risk obstetric or preterm birth clinic.
Arterial catheterisation in neonates can result in severe ischaemic complications but does not impair long‐term extremity function
Authors: Deindl Philipp , Waldhör Thomas , Unterasinger Lukas , Berger Angelika , Keck Maike 
Published: September 2017

AbstractAimWe evaluated the incidence of arterial catheterisation and analysed the risk factors and the extent and outcome of ischaemic complications in neonates.MethodsThis was a retrospective cohort study of 1506 neonates admitted to two, ten‐bed neonatal intensive care units (NICUs) at the Medical University of Vienna, Austria, between 1 January 2011 and 31 December 2014. Medical charts, daily reports and photo documentation were reviewed for arterial catheterisation and ischaemic complications. Patients with severe ischaemic complications were followed up to evaluate extremity function, scaring and cosmetic results.ResultsThere were 542 arterial catheterisations in 485 patients, including 275 born below 28 weeks, which resulted in severe complications in 19 of 485 (4%) patients. Three died before follow‐up. Patients with complications had a significantly lower birthweight, lower postmenstrual age and higher rates of intracranial haemorrhage, retinopathy of prematurity and necrotising enterocolitis. They had also undergone multiple arterial catheterisations more often, needed longer inotropic support and had longer NICU stays than patients without complications. Extremity function was unimpaired in ten of 16 patients with severe ischaemic complications.ConclusionSevere ischaemic complications in neonates following arterial catheterisation were rare events, but could cause devastating damage. Most patients didn't show impaired extremity function at follow‐up.
Chemokine (C‐X‐C motif) ligand 1 is a myokine induced by palmitate and is required for myogenesis in mouse satellite cells
Authors: Masuda S. , Tanaka M. , Inoue T. , Ohue‐Kitano R. , Yamakage H. , Muranaka K. , Kusakabe T. , Shimatsu A. , Hasegawa K. , Satoh‐Asahara N. 
Published: September 2017

AbstractAimThe functional significance of the myokines, cytokines and peptides produced and released by muscle cells has not been fully elucidated. The purpose of this study was to identify a myokine with increased secretion levels in muscle cells due to saturated fatty acids and to examine the role of the identified myokine in the regulation of myogenesis.MethodsHuman primary myotubes and mouse C2C12 myotubes were used to identify the myokine; its secretion was stimulated by palmitate loading. The role of the identified myokine in the regulation of the activation, proliferation, differentiation and self‐renewal was examined in mouse satellite cells (skeletal muscle stem cells).ResultsPalmitate loading promoted the secretion of chemokine (C‐X‐C motif) ligand 1 (CXCL1) in human primary myotubes, and it also increased CXCL1 gene expression level in C2C12 myotubes in a dose‐ and time‐dependent manner. Palmitate loading increased the production of reactive oxygen species along with the activation of nuclear factor‐kappa B (NF‐κB) signalling. Pharmacological inhibition of NF‐κB signalling attenuated the increase in CXCL1 gene expression induced by palmitate and hydrogen peroxide. Palmitate loading significantly increased CXC receptor 2 gene expression in undifferentiated cells. CXCL1 knockdown attenuated proliferation and myotube formation by satellite cells, with reduced self‐renewal. CXCL1 knockdown also significantly decreased the Notch intracellular domain protein level.ConclusionThese results suggest that secretion of the myokine CXCL1 is stimulated by saturated fatty acids and that CXCL1 promotes myogenesis from satellite cells to maintain skeletal muscle homeostasis.
Epidemiological characterization of a nosocomial outbreak of extended spectrum β‐lactamase Escherichia coli ST‐131 confirms the clinical value of core genome multilocus sequence typing
Authors: Woksepp Hanna , Ryberg Anna , Berglind Linda , Schön Thomas , Söderman Jan 
Published: September 2017

Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST‐131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST‐131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability.
Impact of pain and remission in the functioning of patients with depression in Mainland China, Taiwan, and Hong Kong
Authors: Novick Diego , Shi Qiang , Yue Li , Moneta Maria Victoria , Siddi Sara , Haro Josep Maria 
Published: September 2017

AbstractIntroductionThe present study analyzes functioning during the course of treatment of a major depressive disorder in Mainland China, Taiwan, and Hong Kong.MethodsData in this post hoc analysis were taken from a 24‐week prospective, observational study in 12 countries worldwide. Of these, 422 patients were included from Mainland China (N = 205; 48.6%), Taiwan (N = 199; 47.2%), and Hong Kong (N = 18; 4.2%). Functioning was measured with the Sheehan Disability Scale, pain with the Somatic Symptom Inventory, and depression severity with the Quick Inventory of Depressive Symptomatology Self Report 16. Patients were classified as having no pain, persistent pain, or remitted pain. A mixed model with repeated measures was fitted to analyze the relationship between pain and functioning, adjusting for severity and other factors.ResultsAt baseline, 40% of the patients had painful physical symptoms. At 24 weeks, 6% of the patients had persistent pain. Sixty percent of the patients achieved remission. Patients with pain had a higher severity of depression score and lower functioning (P < .05) at baseline. At 24 weeks, patients with persistent pain had lower functioning (P < .05). The regression model found that clinical remission was associated with higher functioning at endpoint and that patients with persistent pain had lower functioning at endpoint when compared with the no‐pain group.ConclusionsPatients presenting with pain had lower functioning at baseline. At 24 weeks, pain persistence was associated with significantly lower functioning as measured by the Sheehan Disability Scale. Clinical remission was associated with better functional outcomes. The course of pain was related to achieving remission.
Survival after treatment with curative intent for hepatocellular carcinoma among patients with vs without non‐alcoholic fatty liver disease
Authors: Wong C. R. , Njei B. , Nguyen M. H. , Nguyen A. , Lim J. K. 
Published: September 2017

AbstractBackgroundNon‐alcoholic fatty liver disease (NAFLD) is expected to become a leading aetiology of hepatocellular carcinoma (HCC)‐related mortality in the United States. HCC treatments with curative intent (OLT, orthotopic liver transplantation; resection; RFA, radiofrequency ablation) can improve survival in carefully selected patients.AimTo compare survival after receipt of curative treatment for NAFLD and non‐NAFLD‐HCC aetiologies (HCV, chronic hepatitis C; HBV, chronic hepatitis B; ALD, alcoholic liver disease) and by treatment was performed.MethodsA cohort of 17 664 patients was assembled using linked Surveillance, Epidemiology, and End Results and Medicare data from 1991 to 2011 with confirmed diagnosis of HCC.ResultsThe cohort was mostly male, aged 70 (21‐106) years, without cardiovascular disease, and had liver cirrhosis without decompensation, metastatic HCC or large tumour size (>5 cm). The NAFLD‐HCC group was mostly female and older with more cardiovascular disease, metastatic HCC, and large tumour size and less cirrhosis and decompensated liver disease than the non‐NAFLD‐HCC groups. The NAFLD group was 47% less likely to receive any curative treatment as compared with non‐NAFLD aetiologies (OR 0.53, P < .001). NAFLD‐HCC had worse median survival after OLT (3.2, 0‐12.9 years, P = .01) but had improved survival after resection (2.4, 0‐12.0 years, P < .001) as compared with non‐NAFLD‐HCC. No significant survival differences existed for RFA by HCC aetiology. NAFLD was not an independent predictor of mortality after OLT, resection or RFA.ConclusionPatients with NAFLD‐HCC had worse survival after OLT but favourable survival after resection, particularly in the absence of cirrhosis, as compared with non‐NAFLD‐HCC aetiologies.
Effects of illite supplementation on in vitro and in vivo rumen fermentation, microbial population and methane emission of Hanwoo steers fed high concentrate diets
Authors: Biswas Ashraf A. , Lee Sung‐Sill , Mamuad Lovelia L. , Kim Seon‐Ho , Choi Yeon‐Jae , Lee Chanhee , Lee Kichoon , Bae Gui‐Seck , Lee Sang‐Suk 
Published: September 2017

AbstractThis study was conducted to evaluate the effects of feeding supplemental illite to Hanwoo steers on methane (CH4) emission and rumen fermentation parameters. An in vitro ruminal fermentation technique was conducted using a commercial concentrate as substrate and illite was added at different concentrations as treatments: 0%, 0.5%, 1.0%, and 2.0% illite. Total volatile fatty acids (VFA) were different (P < 0.05) at 24 h of incubation where the highest total VFA was observed at 1.0% of illite. Conversely, lowest CH4 production (P < 0.01) was found at 1.0% of illite. In the in vivo experiment, two diets were provided, without illite and with addition of 1% illite. An automated head chamber (GreenFeed) system was used to measure enteric CH4 production. Cattle received illite supplemented feed increased (P < 0.05) total VFA concentrations in the rumen compared with those fed control. Feeding illite numerically decreased CH4 production (g/day) and yield (g/kg dry matter intake). Rumen microbial population analysis indicated that the population of total bacteria, protozoa and methanogens were lower (P < 0.05) for illite compared with the control. Accordingly, overall results suggested that feeding a diet supplemented with 1% illite can have positive effects on feed fermentation in the rumen and enteric CH4 mitigation in beef cattle.