bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstructing foot-and-mouth disease outbreaks: a methods comparison of transmission network models
Authors: Firestone Simon M. , Hayama Yoko , Bradhurst Richard , Yamamoto Takehisa , Tsutsui Toshiyuki , Stevenson Mark A.
Published: March 2019

A number of transmission network models are available that combine genomic and epidemiological data to reconstruct networks of who infected whom during infectious disease outbreaks. For such models to reliably inform decision-making they must be transparently validated, robust, and capable of producing accurate predictions within the short data collection and inference timeframes typical of outbreak responses. A lack of transparent multi-model comparisons reduces confidence in the accuracy of transmission network model outputs, negatively impacting on their more widespread use as decision-support tools. We undertook a formal comparison of the performance of nine published transmission network models based on a set of foot-and-mouth disease outbreaks simulated in a previously free country, with corresponding simulated phylogenies and genomic samples from animals on infected premises. Of the transmission network models tested, Lau’s systematic Bayesian integration framework was found to be the most accurate for inferring the transmission network and timing of exposures, correctly identifying the source of 73% of the infected premises (with 91% accuracy for sources with model support >0.80). The Structured COalescent Transmission Tree Inference provided the most accurate inference of molecular clock rates. This validation study points to which models might be reliably used to reconstruct similar future outbreaks and how to interpret the outputs to inform control. Further research could involve extending the best-performing models to explicitly represent within-host diversity so they can handle next-generation sequencing data, incorporating additional animal and farm-level covariates and combining predictions using Ensemble methods and other approaches.
Linking social preferences and ocean acidification impacts in mussel aquaculture
Authors: Martin Valeska A. San , Gelcich Stefan , Vásquez Lavín Felipe , Ponce Oliva Roberto D. , Hernández José I. , Lagos Nelson A. , Birchenough Silvana N. R. , Vargas Cristian A.
Published: March 2019

Ocean Acidification (OA) has become one of the most studied global stressors in marine science during the last fifteen years. Despite the variety of studies on the biological effects of OA with marine commercial species, estimations of these impacts over consumers’ preferences have not been studied in detail, compromising our ability to undertake an assessment of market and economic impacts resulting from OA at local scales. Here, we use a novel and interdisciplinary approach to fill this gap. We experimentally test the impact of OA on commercially relevant physical and nutritional attributes of mussels, and then we use economic discrete choice models to assess the marginal effects of these impacts over consumers’ preferences and wellbeing. Results showed that attributes, which were significantly affected by OA, are also those preferred by consumers. Consumers are willing to pay on average 52% less for mussels with evidences of OA and are willing to increase the price they pay to avoid negative changes in attributes due to OA. The interdisciplinary approach developed here, complements research conducted on OA by effectively informing how OA economic impacts can be analyzed under the lens of marginal changes in market price and consumer’ welfare. Thereby, linking global phenomena to consumers’ wellbeing, and shifting the focus of OA impacts to assess the effects of local vulnerabilities in a wider context of people and businesses.
Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs
Authors: Okamoto Sachiko , Amaishi Yasunori , Maki Izumi , Enoki Tatsuji , Mineno Junichi
Published: March 2019

Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.
p5RHH nanoparticle-mediated delivery of AXL siRNA inhibits metastasis of ovarian and uterine cancer cells in mouse xenografts
Authors: Mills Kathryn A. , Quinn Jeanne M. , Roach S. Tanner , Palisoul Marguerite , Nguyen Mai , Noia Hollie , Guo Lei , Fazal Jawad , Mutch David G. , Wickline Samuel A. , Pan Hua , Fuh Katherine C.
Published: March 2019

Ovarian and uterine serous cancers are extremely lethal diseases that often present at an advanced stage. The late-stage diagnosis of these patients results in the metastasis of their cancers throughout the peritoneal cavity leading to death. Improving survival for these patients will require identifying therapeutic targets, strategies to target them, and means to deliver therapies to the tumors. One therapeutic target is the protein AXL, which has been shown to be involved in metastasis in both ovarian and uterine cancer. An effective way to target AXL is to silence its expression with small interfering RNA (siRNA). We investigate the ability of the novel siRNA delivery platform, p5RHH, to deliver anti-AXL siRNA (siAXL) to tumor cells both in vitro and in vivo as well as examine the phenotypic effects of this siRNA interference. First, we present in vitro assays showing p5RHH-siAXL treatment reduces invasion and migration ability of ovarian and uterine cancer cells. Second, we show p5RHH nanoparticles target to tumor cells in vivo. Finally, we demonstrate p5RHH-siAXL treatment reduces metastasis in a uterine cancer mouse xenograft model, without causing an obvious toxicity. Collectively, these findings suggest that this novel therapy shows promise in the treatment of ovarian and uterine cancer patients.
Hydroxyl radicals generated by hydrogen peroxide photolysis recondition biofilm-contaminated titanium surfaces for subsequent osteoblastic cell proliferation
Authors: Nakamura Keisuke , Shirato Midori , Tenkumo Taichi , Kanno Taro , Westerlund Anna , Örtengren Ulf , Sasaki Keiichi , Niwano Yoshimi
Published: March 2019

Titanium dental implants have been successfully used for decades; however, some implants are affected by peri-implantitis due to bacterial infection, resulting in loss of supporting bone. This study aimed to evaluate the effect of an antimicrobial chemotherapy employing H2O2 photolysis—developed to treat peri-implantitis—on biofilm-contaminated titanium surfaces in association with osteoblastic cell proliferation on the treated surface. Titanium discs were sandblasted and acid-etched, followed by contamination with a three-species biofilm composed of Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mitis. This biofilm model was used as a simplified model of clinical peri-implantitis biofilm. The discs were subjected to ultrasound scaling, followed by H2O2 photolysis, wherein 365-nm LED irradiation of the disc immersed in 3% H2O2 was performed for 5 min. We analysed proliferation of mouse osteoblastic cells (MC3T3-E1) cultured on the treated discs. Compared with intact discs, biofilm contamination lowered cell proliferation on the specimen surface, whereas H2O2 photolysis recovered cell proliferation. Thus, H2O2 photolysis can recover the degraded biocompatibility of biofilm-contaminated titanium surfaces and can potentially be utilised for peri-implantitis treatment. However, to verify the findings of this study in relation to clinical settings, assessment using a more clinically relevant multi-species biofilm model is necessary.
Probiotic Lactobacillus rhamnosus GR-1 is a unique prophylactic agent that suppresses infection-induced myometrial cell responses
Authors: Kim Bona , Shynlova Oksana , Lye Stephen
Published: March 2019

Preterm birth (PTB) is a multifactorial syndrome affecting millions of neonates worldwide. Intrauterine infection can induce PTB through the secretion of pro-inflammatory cytokines and untimely activation of uterine contractions. In pregnant mice, prophylactic administration of probioticLactobacillus rhamnosus GR-1supernatant (GR1SN) prevented lipopolysaccharide (LPS)-induced PTB and reduced cytokine expression in the uterine muscle (myometrium). In this study we sought to delineate the mechanisms by which GR1SN suppressed cytokine secretion in the myometrium. We observed thatL.rhamnosus GR-1uniquely secretes heat-resistant but trypsin-sensitive factors, which significantly suppressed LPS-induced secretion of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in the human myometrial cell line, hTERT-HM. This effect was unique to GR1SN and could not be replicated using supernatant derived from non-GR-1commensal lactobacilli species:L.rhamnosus GG,L.lactis,L.casei, orL.reuteri RC-14. Furthermore, pre-incubation of hTERT-HM cells with low-dose Pam3CSK (a TLR1/2 synthetic agonist which mimics LPS action) prior to LPS administration also significantly decreased LPS-induced cytokine secretion. This study highlights the distinct capacity of protein-like moieties secreted byL.rhamnosus GR-1to inhibit pro-inflammatory cytokine production by human myometrial cells, potentially through a TLR1/2-mediated mechanism.
A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads
Authors: Choi Youngeun , Schmidt Carsten , Tinnefeld Philip , Bald Ilko , Rödiger Stefan
Published: March 2019

The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.
Dirac gap opening and Dirac-fermion-mediated magnetic coupling in antiferromagnetic Gd-doped topological insulators and their manipulation by synchrotron radiation
Authors: Shikin A. M. , Estyunin D. A. , Surnin Yu. I. , Koroleva A. V. , Shevchenko E. V. , Kokh K. A. , Tereshchenko O. E. , Kumar S. , Schwier E. F. , Shimada K. , Yoshikawa T. , Saitoh Y. , Takeda Y. , Kimura A.
Published: March 2019

A new kind of magnetically-doped antiferromagnetic (AFM) topological insulators (TIs) with stoichiometry Bi1.09Gd0.06Sb0.85Te3 has been studied by angle-resolved photoemission spectroscopy (ARPES), superconducting magnetometry (SQUID) and X-ray magnetic circular dichroism (XMCD) with analysis of its electronic structure and surface-derived magnetic properties at different temperatures. This TI is characterized by the location of the Dirac gap at the Fermi level (EF) and a bulk AFM coupling below the Neel temperature (4–8 K). At temperatures higher than the bulk AFM/PM transition, a surface magnetic layer is proposed to develop, where the coupling between the magnetic moments located at magnetic impurities (Gd) is mediated by the Topological Surface State (TSS) via surface Dirac-fermion-mediated magnetic coupling. This hypothesis is supported by a gap opening at the Dirac point (DP) indicated by the surface-sensitive ARPES, a weak hysteresis loop measured by SQUID at temperatures between 30 and 100 K, XMCD measurements demonstrating a surface magnetic moment at 70 K and a temperature dependence of the electrical resistance exhibiting a mid-gap semiconducting behavior up to temperatures of 100–130 K, which correlates with the temperature dependence of the surface magnetization and confirms the conclusion that only TSS are located at the EF. The increase of the TSS’s spectral weight during resonant ARPES at a photon energy corresponding to the Gd 4d-4f edge support the hypothesis of a magnetic coupling between the Gd ions via the TSS and corresponding magnetic moment transfer at elevated temperatures. Finally, the observed out-of-plane and in-plane magnetization induced by synchrotron radiation (SR) due to non-equal depopulation of the TSS with opposite momentum, as seen through change in the Dirac gap value and the k∥-shift of the Dirac cone (DC) states, can be an indicator of the modification of the surface magnetic coupling mediated by the TSS.
The polyadenylation inhibitor cordycepin reduces pain, inflammation and joint pathology in rodent models of osteoarthritis
Authors: Ashraf Sadaf , Radhi Masar , Gowler Peter , Burston James J. , Gandhi Raj D. , Thorn Graeme J. , Piccinini Anna M. , Walsh David A. , Chapman Victoria , de Moor Cornelia H.
Published: March 2019

Clinically, osteoarthritis (OA) pain is significantly associated with synovial inflammation. Identification of the mechanisms driving inflammation could reveal new targets to relieve this prevalent pain state. Herein, a role of polyadenylation in OA synovial samples was investigated, and the potential of the polyadenylation inhibitor cordycepin (3’ deoxyadenosine) to inhibit inflammation as well as to reduce pain and structural OA progression were studied. Joint tissues from people with OA with high or low grade inflammation and non-arthritic post-mortem controls were analysed for the polyadenylation factor CPSF4 and inflammatory markers. Effects of cordycepin on pain behavior and joint pathology were studied in models of OA (intra-articular injection of monosodium iodoacetate in rats and surgical destabilisation of the medial meniscus in mice). Human monocyte-derived macrophages and a mouse macrophage cell line were used to determine effects of cordycepin on nuclear localisation of the inflammatory transcription factor NFĸB and polyadenylation factors (WDR33 and CPSF4). CPSF4 and NFκB expression were increased in synovia from OA patients with high grade inflammation. Cordycepin reduced pain behaviour, synovial inflammation and joint pathology in both OA models. Stimulation of macrophages induced nuclear localisation of NFĸB and polyadenylation factors, effects inhibited by cordycepin. Knockdown of polyadenylation factors also prevented nuclear localisation of NFĸB. The increased expression of polyadenylation factors in OA synovia indicates a new target for analgesia treatments. This is supported by the finding that polyadenylation factors are required for inflammation in macrophages and by the fact that the polyadenylation inhibitor cordycepin attenuates pain and pathology in models of OA.
Molecular profiling of resident and infiltrating mononuclear phagocytes during rapid adult retinal degeneration using single-cell RNA sequencing
Authors: Ronning Kaitryn E. , Karlen Sarah J. , Miller Eric B. , Burns Marie E.
Published: March 2019

Neuroinflammation commonly accompanies neurodegeneration, but the specific roles of resident and infiltrating immune cells during degeneration remains controversial. Much of the difficulty in assessing myeloid cell-specific functions during disease progression arises from the inability to clearly distinguish between activated microglia and bone marrow-derived monocytes and macrophages in various stages of differentiation and activation within the central nervous system. Using an inducible model of photoreceptor cell death, we investigated the prevalence of infiltrating monocytes and macrophage subpopulations after the initiation of degeneration in the mouse retina. In vivo retinal imaging revealed infiltration of CCR2+ leukocytes across retinal vessels and into the parenchyma within 48 hours of photoreceptor degeneration. Immunohistochemistry and flow cytometry confirmed and characterized these leukocytes as CD11b+CD45+ cells. Single-cell mRNA sequencing of the entire CD11b+CD45+ population revealed the presence of resting microglia, activated microglia, monocytes, and macrophages as well as 12 distinct subpopulations within these four major cell classes. Our results demonstrate a previously immeasurable degree of molecular heterogeneity in the innate immune response to cell-autonomous degeneration within the central nervous system and highlight the necessity of unbiased high-throughput and high-dimensional molecular techniques like scRNAseq to understand the complex and changing landscape of immune responders during disease progression.
A substitute variety for agronomically and medicinally important Serenoa repens (saw palmetto)
Authors: Jaiswal Yogini , Weber Daniel , Yerke Aaron , Xue Yanling , Lehman Danielle , Williams Taufika , Xiao Tiqiao , Haddad Daniel , Williams Leonard
Published: March 2019

Serenoa repens (saw palmetto) berries are one of the most consumed medicinal herbs in the United States and the wild green variety is used in the initial therapy of benign prostatic hyperplasia (BPH), globally. Use of saw palmetto is approved by the German Commission E, and several clinical trials are underway for evaluation of its efficacy. Exploitation of its habitats and over foraging imperil this plant, which only grows in the wild. This is the first study, to propose the use of the S. repens forma glauca (silver variety) as a qualitative substitute for the wild variety, to support its conservation. We compared tissue microstructures and lipid and water distribution through spatial imaging and examined metabolite distribution of three tissue domains and whole berries. This combined approach of 3D imaging and metabolomics provides a new strategy for studying phenotypic traits and metabolite synthesis of closely related plant varieties.
Incidence of and Risk Factors for Complex Regional Pain Syndrome Type 1 after Surgery for Distal Radius Fractures: A Population-based Study
Authors: Jo Young-Hoon , Kim KangWook , Lee Bong-Gun , Kim Joo-Hak , Lee Chang-Hun , Lee Kwang-Hyun
Published: March 2019

This study aimed to evaluate the incidence rates of and risk factors for complex regional pain syndrome type 1 (CRPS-1) after surgery for distal radius fractures (DRFs). Using data from January 2007 to December 2014, we analysed the data from the Korean Health Insurance Review and Assessment (HIRA) service. After extracting the data of patients aged ≥18 years whose diagnostic and operation codes for DRFs were entered into the HIRA database, we analysed the incidence rates of and risk factors for CRPS-1. From 2007 to 2014, 172,194 DRFs were treated surgically. Within 1 year postoperatively, 1,103 CRPS-1 cases were diagnosed, with an incidence of 0.64%. On univariate and multivariate analyses, the risk factors that significantly correlated with the incidence of CRPS-1 included female sex, rheumatoid arthritis, open reduction, open fracture, and accompanying ulnar fracture, whereas old age, psychiatric disease, and external fixation were not statistically significant. The incidence of CRPS-1 after surgery for DRF was very low (0.64%) in South Korea. Careful monitoring is necessary for patients with complex fractures and rheumatoid arthritis who are at increased risk of developing CRPS-1.
Anatomic Conformation of Renal Sympathetic Nerve Fibers in Living Human Tissues
Authors: Choe Won-Seok , Song Won Hoon , Jeong Chang Wook , Choi Eue-Keun , Oh Seil
Published: March 2019

Renal denervation using radiofrequency catheter ablation is known to eliminate the renal sympathetic nerve and to lower blood pressure in patients with resistant hypertension. We sought to investigate the detailed anatomic conformation of the peri-renal arterial sympathetic nerve fibers with living human specimens. Peri-renal arterial tissue was harvested from patients undergoing elective radical or simple nephrectomy. Digital images of each section from the distal arterial bifurcation to the proximal margin were obtained and analyzed after immunohistochemical staining with anti-tyrosine hydroxylase antibodies. A total of 3,075 nerve fibers were identified from 84 sections of peri-renal arterial tissue from 28 patients (mean age 62.5 ± 10.2 years, male 68%). Overall, 16% of nerve fibers were located at distances greater than 3 mm from the endoluminal surface of the renal artery. The median distance from the arterial lumen to the nerve fibers of the proximal, middle, and distal renal arterial segments was 1.51 mm, 1.48 mm, and 1.52 mm, respectively. The median diameter of the nerve fibers was 65 μm, and there was no significant difference between the segments. A substantial proportion of the sympathetic nerve fibers were located deeper in the peri-arterial soft tissue than in the lesion depth created by the conventional catheter-based renal sympathetic denervation system.
Potential involvement of Helicobacter pylori from oral specimens in overweight body-mass index
Authors: Hamada Masakazu , Nomura Ryota , Ogaya Yuko , Matayoshi Saaya , Kadota Tamami , Morita Yumiko , Uzawa Narikazu , Nakano Kazuhiko
Published: March 2019

The bacterium Helicobacter pylori was originally classified in the Campylobacter genus, which contains major periodontopathic bacterial species, and H. pylori DNA has been found in the oral cavity. Although many studies show an association between the presence of periodontal bacteria and an overweight body-mass index (BMI; >25 kg/m2), the relationship between body weight and the presence of H. pylori in the oral cavity has not been demonstrated. Herein, we analysed the relationship between H. pylori in the oral cavity and systemic conditions, including the overweight BMI. Saliva specimens and extracted teeth were obtained from 87 subjects; the distribution of H. pylori among these specimens was analysed with the polymerase chain reaction. Subjects with an overweight BMI exhibited significantly higher detection rates of H. pylori in saliva, compared with non-overweight subjects (BMI <25 kg/m2) (P < 0.05). A clinical history of digestive diseases was not associated with the presence of H. pylori in overweight subjects, whereas subjects with both severe dental caries and an overweight BMI showed a higher detection rate of H. pylori in saliva specimens, compared with other groups. These results suggest that the detection of H. pylori in the oral cavity could be associated with the overweight BMI, which was predominant among subjects with severe dental caries.
Ligand entry in human ileal bile acid-binding protein is mediated by histidine protonation
Authors: Horváth Gergő , Egyed Orsolya , Tang Changguo , Kovács Mihály , Micsonai András , Kardos József , Toke Orsolya
Published: March 2019

Human ileal bile acid-binding protein (hI-BABP) has a key role in the intracellular transport of bile salts. To explore the role of histidine protonation in the binding process, the pH-dependence of bile salt binding and internal dynamics in hI-BABP was investigated using NMR spectroscopy and biophysical tools. Thermodynamic and kinetic measurements show an increase in the overall binding affinity and the association rate constant of the first binding step below the pKaof the histidines, suggesting that ligand binding is favoured by the protonated state. The overlap between residues exhibiting a high sensitivity to pH in their backbone amide chemical shifts and protein regions undergoing a global ms conformational exchange indicate a connection between the two processes. According to15N NMR relaxation dispersion analysis, the slow motion is most pronounced at and above the pKaof the histidines. In agreement with the NMR measurements, MD simulations show a stabilization of the protein by histidine protonation. Hydrogen-bonding and van der Waals interactions mediating the flow of information between the C/D- and G/H-turn regions hosting the three histidines, suggest a complex way of pH-governed allosteric regulation of ligand entry involving a transition between a closed and a more open protein state.
Photonic jet lens
Authors: Lecler Sylvain , Perrin Stephane , Leong-Hoi Audrey , Montgomery Paul
Published: March 2019

Microsphere-assisted microscopy currently benefits from a considerable interest in the microscope-research community. Indeed, this new imaging technique enables the lateral resolution of optical microscopes to reach around λ/5 through a full-field and a far-field acquisition while being label-free. Despite the photonic jet clearly not being a relevant concept to justify the super-resolution phenomenon, we show here how it can be used to predict imaging formation and performance such as the image position and the microsphere magnification. This study allows a better understanding of the experimental measurements that have been observed over the last decade and that will be observed in coming years, through numerical simulations using different optical and geometrical parameters.
The formation mechanism of Li4Ti5O12−y solid solutions prepared by carbothermal reduction and the effect of Ti3+ on electrochemical performance
Authors: Yang Guijun , Park Soo-Jin
Published: March 2019

Samples of Li4Ti5O12−y solid solutions are synthesized by one-step solid-state carbothermal reduction reaction using Li2CO3, anatase, and carbon black under a nitrogen atmosphere. The underlying formation mechanism that leads to Li4Ti5O12−y solid solutions is proposed. The formation mechanism of the Li4Ti5O12−y solid solution is investigated by in situ variable temperature X-Ray diffraction (VT-XRD) and thermogravimetric analysis/differential scanning calorimetry (TGA-DSC). First, some Ti4+ centers are converted to Ti3+ (TiO2-TiO2−x) because of the presence of carbon black. Secondly, Li2CO3 reacts with TiO2−x (anatase) to form Li2TiO3. Thirdly, Li2TiO3 reacts with TiO2−x to form the Li4Ti5O12−y solid solution, while anatase starts to transform into rutile at the same time. Rutile reacts with Li2TiO3 to form Li4Ti5O12−y at higher temperatures. The presence of Ti3+ not only improves the electrical conductivity but also improves the ionic conductivity. As a result, the as-prepared material exhibits good rate capability and cycling stability with 99.3% capacity retention after 200 cycles.
Targeted Single Primer Enrichment Sequencing with Single End Duplex-UMI
Authors: Peng Quan , Xu Chang , Kim Daniel , Lewis Marcus , DiCarlo John , Wang Yexun
Published: March 2019

For specific detection of somatic variants at very low levels, artifacts from the NGS workflow have to be eliminated. Various approaches using unique molecular identifiers (UMI) to analytically remove NGS artifacts have been described. Among them, Duplex-seq was shown to be highly effective, by leveraging the sequence complementarity of two DNA strands. However, all of the published Duplex-seq implementations so far required pair-end sequencing and in the case of combining duplex sequencing with target enrichment, lengthy hybridization enrichment was required. We developed a simple protocol, which enabled the retrieval of duplex UMI in multiplex PCR based enrichment and sequencing. Using this protocol and reference materials, we demonstrated the accurate detection of known SNVs at 0.1–0.2% allele fractions, aided by duplex UMI. We also observed that low level base substitution artifacts could be introduced when preparing in vitro DNA reference materials, which could limit their utility as a benchmarking tool for variant detection at very low levels. Our new targeted sequencing method offers the benefit of using duplex UMI to remove NGS artifacts in a much more simplified workflow than existing targeted duplex sequencing methods.
Urine-NMR metabolomics for screening of advanced colorectal adenoma and early stage colorectal cancer
Authors: Kim Eun Ran , Kwon Hyuk Nam , Nam Hoonsik , Kim Jae J. , Park Sunghyouk , Kim Young-Ho
Published: March 2019

Although colorectal cancer (CRC) is considered one of the most preventable cancers, no non-invasive, accurate diagnostic tool to screen CRC exists. We explored the potential of urine nuclear magnetic resonance (NMR) metabolomics as a diagnostic tool for early detection of CRC, focusing on advanced adenoma and stage 0 CRC. Urine metabolomics profiles from patients with colorectal neoplasia (CRN; 36 advanced adenomas and 56 CRCs at various stages, n = 92) and healthy controls (normal, n = 156) were analyzed by NMR spectroscopy. Healthy and CRN groups were statistically discriminated using orthogonal projections to latent structure discriminant analysis (OPLS-DA). The class prediction model was validated by three-fold cross-validation. The advanced adenoma and stage 0 CRC were grouped together as pre-invasive CRN. The OPLS-DA score plot showed statistically significant discrimination between pre-invasive CRN as well as advanced CRC and healthy controls with a Q2 value of 0.746. In the prediction validation study, the sensitivity and specificity for diagnosing pre-invasive CRN were 96.2% and 95%, respectively. The grades predicted by the OPLS-DA model showed that the areas under the curve were 0.823 for taurine, 0.783 for alanine, and 0.842 for 3-aminoisobutyrate. In multiple receiver operating characteristics curve analyses, taurine, alanine, and 3-aminoisobutyrate were good discriminators for CRC patients. NMR-based urine metabolomics profiles significantly and accurately discriminate patients with pre-invasive CRN as well as advanced CRC from healthy individuals. Urine-NMR metabolomics has potential as a screening tool for accurate diagnosis of pre-invasive CRN.
Near real-time enumeration of live and dead bacteria using a fibre-based spectroscopic device
Authors: Ou Fang , McGoverin Cushla , Swift Simon , Vanholsbeeck Frédérique
Published: March 2019

A rapid, cost-effective and easy method that allows on-site determination of the concentration of live and dead bacterial cells using a fibre-based spectroscopic device (the optrode system) is proposed and demonstrated. Identification of live and dead bacteria was achieved by using the commercially available dyes SYTO 9 and propidium iodide, and fluorescence spectra were measured by the optrode. Three spectral processing methods were evaluated for their effectiveness in predicting the original bacterial concentration in the samples: principal components regression (PCR), partial least squares regression (PLSR) and support vector regression (SVR). Without any sample pre-concentration, PCR achieved the most reliable results. It was able to quantify live bacteria from 108 down to 106.2 bacteria/mL and showed the potential to detect as low as 105.7 bacteria/mL. Meanwhile, enumeration of dead bacteria using PCR was achieved between 108 and 107 bacteria/mL. The general procedures described in this article can be applied or modified for the enumeration of bacteria within populations stained with fluorescent dyes. The optrode is a promising device for the enumeration of live and dead bacterial populations particularly where rapid, on-site measurement and analysis is required.
Diversity of incubation rhythms in a facultatively uniparental shorebird – the Northern Lapwing
Authors: Sládeček Martin , Vozabulová Eva , Šálek Miroslav E. , Bulla Martin
Published: March 2019

In birds, incubation by both parents is a common form of care for eggs. Although the involvement of the two parents may vary dramatically between and within pairs, as well as over the course of the day and breeding season, detailed descriptions of this variation are rare, especially in species with variable male contributions to care. Here, we continuously video-monitored 113 nests of Northern LapwingsVanellus vanellusto reveal the diversity of incubation rhythms and parental involvement, as well as their daily and seasonal variation. We found great between-nest variation in the overall nest attendance (68–94%; median = 87%) and in how much males attended their nests (0–37%; median = 13%). Notably, the less the males attended their nests, the lower was the overall nest attendance, even though females partially compensated for the males’ decrease. Also, despite seasonal environmental trends (e.g. increasing temperature), incubation rhythms changed little over the season and 27-day incubation period. However, as nights shortened with the progressing breeding season, the longest night incubation bout of females shortened too. Importantly, within the 24h-day, nest attendance was highest, incubation bouts longest, exchange gaps shortest and male involvement lowest during the night. Moreover, just after sunrise and before sunset males attended the nest the most. To conclude, we confirm substantial between nest differences in Lapwing male nest attendance, reveal how such differences relates to variation in incubation rhythms, and describe strong circadian incubation rhythms modulated by sunrise and sunset.
Mechanical Response of He-Implanted Amorphous SiOC/Crystalline Fe Nanolaminates
Authors: Zare A. , Su Q. , Gigax J. , Harriman T. A. , Nastasi M. , Shao L. , Lucca D. A.
Published: March 2019

This study investigates the microstructural evolution and mechanical response of sputter-deposited amorphous silicon oxycarbide (SiOC)/crystalline Fe nanolaminates, a single layer SiOC film, and a single layer Fe film subjected to ion implantation at room temperature to obtain a maximum He concentration of 5 at. %. X-ray diffraction and transmission electron microscopy indicated no evidence of implantation-induced phase transformation or layer breakdown in the nanolaminates. Implantation resulted in the formation of He bubbles and an increase in the average size of the Fe grains in the individual Fe layers of the nanolaminates and the single layer Fe film, but the bubble density and grain size were found to be smaller in the former. By reducing the thicknesses of individual layers in the nanolaminates, bubble density and grain size were further decreased. No He bubbles were observed in the SiOC layers of the nanolaminates and the single layer SiOC film. Nanoindentation and scanning probe microscopy revealed an increase in the hardness of both single layer SiOC and Fe films after implantation. For the nanolaminates, changes in hardness were found to depend on the thicknesses of the individual layers, where reducing the layer thickness to 14 nm resulted in mitigation of implantation-induced hardening.
Temporal evolution of quantitative EEG within 3 days of birth in early preterm infants
Authors: O’Toole John M. , Pavlidis Elena , Korotchikova Irina , Boylan Geraldine B. , Stevenson Nathan J.
Published: March 2019

For the premature newborn, little is known about changes in brain activity during transition to extra-uterine life. We aim to quantify these changes in relation to the longer-term maturation of the developing brain. We analysed EEG for up to 72 hours after birth from 28 infants born <32 weeks of gestation. These infants had favourable neurodevelopment at 2 years of age and were without significant neurological compromise at time of EEG monitoring. Quantitative EEG was generated using features representing EEG power, discontinuity, spectral distribution, and inter-hemispheric connectivity. We found rapid changes in cortical activity over the 3 days distinct from slower changes associated with gestational age: for many features, evolution over 1 day after birth is equivalent to approximately 1 to 2.5 weeks of maturation. Considerable changes in the EEG immediately after birth implies that postnatal adaption significantly influences cerebral activity for early preterm infants. Postnatal age, in addition to gestational age, should be considered when analysing preterm EEG within the first few days after birth.
Contribution of time of day and the circadian clock to the heat stress responsive transcriptome in Arabidopsis
Authors: Blair Emily J. , Bonnot Titouan , Hummel Maureen , Hay Erika , Marzolino Jill M. , Quijada Ivan A. , Nagel Dawn H.
Published: March 2019

InArabidopsis, a large subset of heat responsive genes exhibits diurnal or circadian oscillations. However, to what extent the dimension of time and/or the circadian clock contribute to heat stress responses remains largely unknown. To determine the direct contribution of time of day and/or the clock to differential heat stress responses, we probed wild-type and mutants of the circadian clock genesCCA1,LHY,PRR7, andPRR9following exposure to heat (37 °C) and moderate cold (10 °C) in the early morning (ZT1) and afternoon (ZT6). Thousands of genes were differentially expressed in response to temperature, time of day, and/or the clock mutation. Approximately 30% more genes were differentially expressed in the afternoon compared to the morning, and heat stress significantly perturbed the transcriptome. Of the DEGs (~3000) specifically responsive to heat stress, ~70% showed time of day (ZT1 or ZT6) occurrence of the transcriptional response. For the DEGs (~1400) that are shared between ZT1 and ZT6, we observed changes to the magnitude of the transcriptional response. In addition, ~2% of all DEGs showed differential responses to temperature stress in the clock mutants. The findings in this study highlight a significant role for time of day in the heat stress responsive transcriptome, and the clock throughCCA1andLHY, appears to have a more profound role thanPRR7andPRR9in modulating heat stress responses during the day. Our results emphasize the importance of considering the dimension of time in studies on abiotic stress responses inArabidopsis.
Complex regulatory network allows Myriophyllum aquaticum to thrive under high-concentration ammonia toxicity
Authors: Wang Rui , Xu Shengjun , Sun Haishu , Feng Shugeng , Jiang Cancan , Zhou Sining , Wu Shimin , Zhuang Guoqiang , Chen Baodong , Bai Zhihui , Zhuang Xuliang
Published: March 2019

Plants easily experience ammonia (NH4+) toxicity, especially aquatic plants. However, a unique wetland plant species,Myriophyllum aquaticum, can survive in livestock wastewater with more than 26 mM NH4+. In this study, the mechanisms of theM. aquaticumresponse to NH4+toxicity were analysed with RNA-seq. Preliminary analysis of enzyme activities indicated that key enzymes involved in nitrogen metabolism were activated to assimilate toxic NH4+into amino acids and proteins. In response to photosystem damage,M. aquaticumseemed to remobilize starch and cellulose for greater carbon and energy supplies to resist NH4+toxicity. Antioxidative enzyme activity and the secondary metabolite content were significantly elevated for reactive oxygen species removal. Transcriptomic analyses also revealed that genes involved in diverse functions (e.g., nitrogen, carbon and secondary metabolisms) were highly responsive to NH4+stress. These results suggested that a complex physiological and genetic regulatory network inM. aquaticumcontributes to its NH4+tolerance.
Comprehensive analysis of resistance-nodulation-cell division superfamily (RND) efflux pumps from Serratia marcescens, Db10
Authors: Toba Shinsuke , Minato Yusuke , Kondo Yuma , Hoshikawa Kanami , Minagawa Shu , Komaki Shiho , Kumagai Takanori , Matoba Yasuyuki , Morita Daichi , Ogawa Wakano , Gotoh Naomasa , Tsuchiya Tomofusa , Kuroda Teruo
Published: March 2019

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coli. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6 G efflux assay. When expressed in the tolC-deficient strain of E. coli, KAM43, none of the genes conferred resistance on E. coli. When hasF, encoding the S. marcescens TolC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coli, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents.
Revealing transient events of molecular recognition via super-localization imaging of single-particle motion
Authors: Kong Qing-Ying , Yang Fan , Song Juan , Ruan Yi-Fan , Li Shan-Shan , Gao Zhao-Shuai , Kang Bin , Chen Hong-Yuan , Xu Jing-Juan
Published: March 2019

Molecular recognition plays an important role in biological systems and relates to a wide range of applications in disease diagnostics and therapeutics. Studies based on steady state or ensemble analysis may mask critical dynamic information of single recognition events. Here we report a study of monitoring the transient molecular recognition via single particle motion. We utilized a super-localization imaging methodology, to comprehensively evaluate the rotational Brownian motion of a single nanoparticle in spatial-temporal-frequential domain, with a spatial accuracy ~20 nm and a temporal resolution of ~10 ms. The transient moment of molecular encountering was captured and different binding modes were discriminated. We observed that the transient recognition events were not static states of on or off, but stochastically undergoes dynamical transformation between different binding modes. This study improves our understanding about the dynamic nature of molecular recognition events beyond the ensemble characterization via binding constant.
Design principles of biologically fabricated avian nests
Authors: Jessel Hadass R. , Chen Sagi , Osovski Shmuel , Efroni Sol , Rittel Daniel , Bachelet Ido
Published: March 2019

Materials and construction methods of nests vary between bird species and at present, very little is known about the relationships between architecture and function in these structures. This study combines computational and experimental techniques to study the structural biology of nests fabricated by the edible nest swiftlet Aerodramus fuciphagus on vertical rock walls using threaded saliva. Utilizing its own saliva as a construction material allows the swiftlets full control over the structural features at a very high resolution in a process similar to additive manufacturing. It was hypothesized that the mechanical properties would vary between the structural regions of the nest (i.e. anchoring to the wall, center of the cup, and rim) mainly by means of architecture to offer structural support and bear the natural loads of birds and eggs. We generated numerical models of swiftlet nests from μCT scans based on collected swiftlet nests, which we loaded with a force of birds and eggs. This was done in order to study and assess the stress distribution that characterizes the specific nest’s architecture, evaluate its strength and weak points if any, as well as to understand the rationale and benefits that underlie this natural structure. We show that macro- and micro-scale structural patterns are identical in all nests, suggesting that their construction is governed by specific design principles. The nests’ response to applied loads of birds and eggs in finite element simulations suggests a mechanical overdesign strategy, which ensures the stresses experienced by its components in any loading scenario are actively minimized to be significantly smaller than the tensile fracture strength of the nests’ material. These findings highlight mechanical overdesign as a biological strategy for resilient, single-material constructions designed to protect eggs and hatchlings.
A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation
Authors: Saadi Wiam , Kermezli Yasmina , Dao Lan T. M. , Mathieu Evelyne , Santiago-Algarra David , Manosalva Iris , Torres Magali , Belhocine Mohamed , Pradel Lydie , Loriod Beatrice , Aribi Mourad , Puthier Denis , Spicuglia Salvatore
Published: March 2019

Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8-double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion ofRobnr, an inducible lncRNA located downstream of the anti-apoptotic geneBcl2, demonstrated a critical role of theRobnrlocus in the induction ofBcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.
RNAi-Mediated PD-L1 Inhibition for Pancreatic Cancer Immunotherapy
Authors: Yoo Byunghee , Jordan Veronica Clavijo , Sheedy Patrick , Billig Ann-Marie , Ross Alana , Pantazopoulos Pamela , Medarova Zdravka
Published: March 2019

The recent past has seen impressive progress in the treatment of various malignancies using immunotherapy. One of the most promising approaches involves immune checkpoint inhibitors. However, the clinical results with these agents have demonstrated variability in the response. Pancreatic cancer, in particular, has proven resistant to initial immunotherapy approaches. Here, we describe an alternative strategy that relies on combining gemcitabine and a novel programmed death-ligand 1 (PD-L1) inhibitor, termed MN-siPDL1. MN-siPDL1 incorporates small interfering RNA against PD-L1 (siPDL1) conjugated to a magnetic nanocarrier (MN). We show that noninvasive magnetic resonance imaging (MRI) could be used to monitor therapeutic response. Combination therapy consisting of gemcitabine and MN-siPDL1 in a syngeneic murine pancreatic cancer model resulted in a significant reduction in tumor growth and an increase in survival. Following optimization, a 90% reduction in tumor volume was achieved 2 weeks after the beginning of treatment. Whereas 100% of the control animals had succumbed to their tumors by week 6 after the beginning of treatment, there was no mortality in the experimental group by week 5, and 67% of the experimental animals survived for 12 weeks. This method could provide therapeutic benefit against an intractable disease for which there are no effective treatments and which is characterized by a mere 1% 5-year survival.