bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry
Authors: Onukwufor John O. , Trewin Adam J. , Baran Timothy M. , Almast Anmol , Foster Thomas H. , Wojtovich Andrew P. 
Published: September 2019

ABSTRACTFluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2•-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2•--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2•- quantum yield (ΦO2•-) of SuperNova was determined to be 0.00150, KillerRed was 0.00097, and mCherry 0.00120. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O2•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield (Φ1O2) for SuperNova to be 0.0220, KillerRed 0.0076, and mCherry 0.0057. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.GRAPHICAL ABSTRACT
Social isolation impacts late-life socio-cognitive decline in APP/PS1 mice
Authors: Rens Eva , D’Hooge Rudi , Van der Jeugd Ann 
Published: September 2019

AbstractIn this study the effects of social isolation (SI) were investigated in APP/PS1 mice. It was found that SI during adolescence has an impact on anxiogenic behaviour, such that isolated animals tend to explore a threatening environment less than non-isolated animals as assessed with the EPM test, and that this holds for both AD and non-AD mice. While no evidence was found for any differences in short-term memory as assessed by the Y-maze, long-term memory seemed to be affected in a context-dependent manner. Object memory as assessed with the NOR test was affected in APP/PS1 mice compared to WT mice, but this deficit was not induced or influenced by SI. When it comes to social recognition memory however, we found that SI exacerbated the social memory deficit in AD mice, and even induced a deficit in WTs. Associative fear memory as assessed with the PA test suggested that WTs perform better when group housed, and APP/PS1 mice better when socially isolated. The link between isolation and AD, or cognition in general, may be more complex than initially thought. The effect of isolation may not be the same for AD versus non-AD subjects.
Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification
Authors: López-Longarela Barbara , Morrison Emma E. , Tranter John D. , Chahman-Vos Lianne , Léonard Jean-François , Gautier Jean-Charles , Laurent Sébastien , Lartigau Aude , Boitier Eric , Sautier Lucile , Carmona-Saez Pedro , Martorell-Marugan Jordi , Mellanby Richard J. , Pernagallo Salvatore , Ilyine Hugh , Rissin David M. , Duffy David C. , Dear James W. , Díaz-Mochón Juan J. 
Published: September 2019

AbstractCirculating microRNAs are biomarkers reported to be stable and translational across species. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free and amplification-free detection method for measuring miR-122 that has translational utility in context of DILI. We developed a single molecule dynamic chemical labelling (DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probe that contained a reactive amine instead of a nucleotide at a specific position in the sequence. The single molecule DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a fit-for-purpose limit of detection of 1.32 pM. In 192 human serum samples, DCL accurately identified patients at risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P<0.0001). The miR-122 assay also quantified liver injury in rats and dogs. When DCL beads were added to serum, the miR-122 signal was stabilised (no loss of signal after 14 days at room temperature). By contrast, there was substantial degradation of miR-122 in the absence of beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiple miR-122 isomiRs with DILI that were at low concentration or not present in healthy patient serum. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analysing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. In summary, the DCL assay can accurately measure miR-122 directly from serum and plasma to diagnose liver injury in humans and other species, and can overcome important microRNA biomarker analytical and biological challenges.
Lipid droplet biogenesis is driven by liquid-liquid phase separation
Authors: Zoni Valeria , Khaddaj Rasha , Campomanes Pablo , Thiam Abdou Rachid , Schneiter Roger , Vanni Stefano 
Published: September 2019

AbstractCells store energy in the form of neutral lipids packaged into micrometer-sized organelles named lipid droplets (LD). These structures emerge from the endoplasmic reticulum (ER), but their biogenesis remains poorly understood. Using molecular simulations, we found that fat accumulation and LD formation are described by a liquid-liquid phase separation (LLPS) process. Within this framework, we could identify how ER membrane properties modulate LD formation, and we could directly test our computational predictions by combining yeast genetics with fluorescence microscopy. Our data suggest that the specific lipid composition of the ER together with its peculiar physical properties, such as low membrane tension and membrane curvature, promote the packaging of neutral lipids into LD, preventing their accumulation in the ER membrane. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.
Coupling chromatin structure and dynamics by live super-resolution imaging
Authors: Barth R. , Bystricky K. , Shaban H. A. 
Published: September 2019

AbstractChromatin conformation regulates gene expression and thus constant remodeling of chromatin structure is essential to guarantee proper cell function. To gain insight into the spatio-temporal organization of the genome, we employ high-density photo-activated localization microscopy and deep learning to obtain temporally resolved super-resolution images of chromatin in living cells. In combination with high-resolution dense motion reconstruction, we confirm the existence of elongated ~ 45 to 90 nm wide chromatin ‘blobs’. A computational chromatin model suggests that these blobs are dynamically associating chromatin fragments in close physical and genomic proximity and adopt TAD-like interactions in the time-average limit. Experimentally, we found that chromatin exhibits a spatio-temporal correlation over ~ 4 μm in space and tens of seconds in time, while chromatin dynamics are correlated over ~ 6 μm and last 40 s. Notably, chromatin structure and dynamics are closely related, which may constitute a mechanism to grant access to regions with high local chromatin concentration.
Isolation housing exacerbates Alzheimer’s Disease phenotype in aged APP KI mice
Authors: Laroy M , Saito T , Saido TC , D’Hooge R , Van der Jeugd A 
Published: September 2019

AbstractIn January 2018, Britain was the first in the world to adopt a Minister of Loneliness. This illustrates the changing view on loneliness: being lonely is not just a feeling of a lack of companionship, but also a serious health problem. For example, we know that loneliness is as bad as smoking 15 cigarettes a day. Moreover, research has shown that lonely people express higher levels of cortical amyloid. Amyloid burden is an important marker of Alzheimer’s Disease (AD), a chronic neurodegenerative disease and the main cause of dementia worldwide. Together with other findings a link between loneliness, (perceived) social isolation and AD is now undeniable, but it is hard to tell from human studies whether it is the cause or the effect of AD. We need standardized animal studies to answer this question.In an effort to study how social isolation and AD interact, we used APP KI mice bearing human transgenes known to cause AD, and isolated part of the mice in order to mimic loneliness in late-life while part of them remained group-housed. We next looked at the effects of isolation on the behaviour and symptomatology typically present in AD patients to tap cognition.Our study reveals mixed results. Results indicate that at before isolation, at the age of 16 and 24 months, APPNL/NLand APPNL-G-F/NL-G-Fmice do not differ to a significant extent on both the behavioural level. The APPNL-G-F/NL-G-Fdifferentiated slightly worse between the conditioned context and a new context compared to the APPNL/NLmice. However, the difference appeared to be more pronounced after a period of social isolation. Social isolation had distinct effects on the AD-related anxiogenic and dementia-like phenotype. Spatial learning in the MWM task revealed distinct differences between our two models. After isolation APPNL-G-F/NL-G-Fmice used less spatial search strategies, compared to control mice, thus reflecting perseveration and less behavioural flexibility due to the isolation period.
Enhanced maturation of human stem cell derived interneurons by mTOR activation
Authors: Chu Jianhua , Fitzgerald Megan L. , Sehgal Neha , Manley William , Fitzgerald Shane , Naung Harrison , Goldberg Ethan M. , Anderson Stewart A. 
Published: September 2019

SummaryThe use of stem cell derived neurons for cell-based therapies is limited by a protracted maturation. We present a novel approach for accelerating the post-mitotic maturation of human stem cell derived interneurons via the activation of mTOR signaling. Lox sites were placed within PTEN, a key mTOR inhibitor, in a cortical interneuron (CIn) reporter line. Following directed differentiation and purification by FACS, the CIns were exposed to Cre-expressing lentivirus, then transplanted into mouse neocortex or plated onto cultured rat neocortex. Input synaptogenesis and dendritogenesis was greatly enhanced in the PTEN-deleted CIns. Whole-cell recording of the PTEN-deleted CIns in slices of transplanted neocortex revealed multiple indices of enhanced maturation. Finally, we observed similar effects using transient, doxycycline-inducible activation of AKT. We thus present an inducible, reversible approach for accelerating the maturation of human stem cell derived CIns, and to study the influences of this disease-related signaling system in human neurons.
Elevated levels of interleukin-27 in early life compromise protective immunity during neonatal sepsis
Authors: Seman Brittany G. , Vance Jordan K. , Rawson Travis W. , Witt Michelle R. , Huckaby Annalisa B. , Povroznik Jessica M. , Bradford Shelby D. , Barbier Mariette , Robinson Cory M. 
Published: September 2019

ABSTRACTNeonates are at increased risk for bacterial sepsis as a result of immature immunity. We established that the immune suppressive cytokine interleukin (IL)-27 is elevated in early life. In the present work, we hypothesized that increased levels of IL-27 may predispose the neonatal population to more severe infection during sepsis. In a neonatal sepsis model, systemic IL-27 levels continued to rise during infection. Peripheral tissue analysis revealed systemic IL-27 expression, while myeloid cell profiling identified Gr-1 and F4/80-expressing cells as the most abundant producers of IL-27 during infection. Increased IL-27 levels were consistent with increased mortality that was improved in WSX-1-/-mice that lack a functional IL-27 receptor. Infected WSX-1-/-pups exhibited improved weight gain and reduced morbidity. IL-27 signaling in WT mice promoted increased bacterial burdens and systemic inflammation compared to WSX-1-/-neonates. This was consistent with more efficient bacterial killing by Ly6B.2+myeloid cells and macrophages from WSX-1-deficient compared to wild-type neonates. Live animal imaging further supported a more severe and disseminated infection in WT neonates. This is the first report to describe the impact of elevated early life IL-27 on the host response in neonates while also defining the cell and tissue sources of cytokine. IL-27 is frequently associated with suppressed inflammation. In contrast, our findings demonstrate that IL-27 promotes inflammation during neonatal sepsis by directly compromising control of bacteria that drive the inflammatory response. Collectively, our results suggest that IL-27 represents a therapeutic target to limit susceptibility and improve infectious outcomes in neonatal sepsis.IMPORTANCEA number of differences in the neonatal immune response compared with adults have been well described. However, a mechanistic understanding of what needs to be overcome in the neonate to generate a more protective immune response during acute bacterial infection has been limited. The work described here helps fill the gap of what is necessary to overcome in order to achieve improved host response to infection. To further the novelty, IL-27 has not previously been attributed to dysfunction or deficiency in neonatal immunity. Our results enhance the understanding of IL-27 biology in the neonatal population while providing evidence that elevated IL-27 levels limit a protective immune response and are detrimental during neonatal sepsis. Strategies aimed at targeting circulating IL-27 concentrations early in life have the potential to improve control of bacterial infection in neonates.
Interleukin-33 promotes type 1 cytokine expression via p38 MAPK in human natural killer cells
Authors: Ochayon David E. , Ali Ayad , Alarcon Pablo C. , Krishnamurthy Durga , Kottyan Leah C. , Borchers Michael , Waggoner Stephen N. 
Published: September 2019

This study tests the hypothesis that activation of mitogen-activated protein kinase (MAPK) by physiologically-relevant concentrations of interleukin-33 (IL-33) contributes to enhanced cytokine expression by IL-12 stimulated human natural killer (NK) cells. While IL-33 canonically triggers type 2 cytokine responses, this cytokine can also synergize with type 1 cytokines like IL-12 to provoke interferon-gamma (IFN-γ). We show that picogram concentrations of IL-12 and IL-33 are sufficient to promote robust secretion of IFN-γ by human NK cells that greatly exceeds responses to either cytokine alone. Nanogram doses of IL-33, potentially consistent with levels in tissue microenvironments, synergize with IL-12 to induce secretion of additional cytokines, including tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-33-induced activation of the p38 MAPK pathway in human NK cells is crucial for enhanced release of IFN-γ and TNF in response to IL-12. Mechanistically, IL-33-induced p38 MAPK signaling enhances stability ofIFNGtranscripts and triggers ADAM17-mediated cleavage of TNF from the cell surface. These data support our hypothesis and suggest that altered sensitivity of NK cells to IL-12 in the presence of IL-33 may have important consequences in diseases associated with mixed cytokine milieus, like asthma and chronic obstructive pulmonary disease.Graphical Abstract
Boiling Acid Mimics Intracellular Giant Virus Genome Release
Authors: Schrad Jason R. , Abrahão Jônatas S. , Cortines Juliana R. , Parent Kristin N. 
Published: September 2019

SummarySince their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four metastable infection intermediates in Samba virus (lineage AMimiviridae) as visualized by cryo-EM, cryo-ET, and SEM. Each of these four intermediates reflects a stage that occursin vivo. We show that these genome release stages are conserved in other, diverse giant viruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved amongst disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.
Specific detection of Trichophyton rubrum and Trichophyton interdigitale based on loop‐mediated isothermal amplification (LAMP) from onychomycosis specimens
Authors: Watanabe Shinichi , Okubo Akihiro , Miyajima Yoshiharu , Satoh Kazuo , Makimura Koichi 
Published: September 2019

AbstractIn diagnosing onychomycosis, diseases with similar features must be excluded by demonstrating the presence of fungal infection and identifying the fungal species. However, fungal culture of onychomycosis‐derived samples usually takes many weeks to yield species identification results, and is associated with a low successful culture rate. Loop‐mediated isothermal amplification (LAMP) is a highly sensitive and specific molecular biological method that can amplify DNA at a constant temperature, allowing for a simpler testing procedure, shorter detection time and less cost than conventional techniques including quantitative polymerase chain reaction. We have developed a new LAMP method specifically to detect Trichophyton rubrum (T. rubrum) and Trichophyton interdigitale (T. interdigitale), major causative dermatophytes for onychomycosis, and analyzed the correlation between LAMP results and those of the existing fungal culture method for the detection and identification of Trichophyton species from onychomycosis‐derived samples. The results showed that all 59 specimens in which T. rubrum or T. interdigitale was identified by fungal culture also tested positive by LAMP, giving a 100% positivity concordance rate between the two methods. Moreover, all 55 and four specimens in which T. rubrum and T. interdigitale were identified by fungal culture, respectively, also tested positive for each species by LAMP, again giving a 100% species‐identification concordance rate. The high correlation demonstrated between LAMP and fungal culture results in detection and identification of Trichophyton species from onychomycosis‐derived samples suggests high reliability of LAMP as a promising, alternative mycological detection and identification technique which can serve as an alternative to the fungal culture method.
Enacting alcohol realities: gendering practices in Australian studies on ‘alcohol‐related presentations’ to emergency departments
Authors: Moore David , Keane Helen , Duncan Duane 
Published: September 2019

Abstract‘Alcohol‐fuelled violence’ and its prevention has been the subject of recent intense policy debate in Australia, with the content of this debate informed by a surprisingly narrow range of research resources. In particular, given the well‐established relationship between masculinities and violence, the meagre attention paid to the role of gender in alcohol research and policy recommendations stands out as a critical issue. In this article, which draws on recent work in feminist science studies and science and technology studies, we focus on the treatment of gender, alcohol and violence in Australian research on ‘alcohol‐related presentations’ to emergency departments (EDs), analysing this type of research because of its prominence in policy debates. We focus on four types of ‘gendering practice’ through which research genders ‘alcohol‐related presentations’ to EDs: omitting gender from consideration, overlooking clearly gendered data when making gender‐neutral policy recommendations, rendering gender invisible via methodological considerations, and addressing gender in terms of risk and vulnerability. We argue that ED research practices and their policy recommendations reproduce normative understandings of alcohol's effects and of the operations of gender in social arrangements, thereby contributing to the ‘evidence base’ supporting unfair policy responses.
Triaging women with human papillomavirus infection and normal cytology or low‐grade dyskaryosis: evidence from 10‐year follow up of the ARTISTIC trial cohort
Authors: Gilham C , Sargent A , Peto J 
Published: September 2019

ObjectivesTo estimate long‐term cervical intraepithelial neoplasia grade 3 (CIN3) risks associated with different triage strategies for human papillomavirus positive (HPV+) women with a view to reducing unnecessary referrals.DesignThe ARTISTIC trial cohort was recruited in Manchester in 2001–03 and was followed up for CIN3 and cancer notification through national registration until December 2015.ResultsThe 10‐year cumulative risk of CIN3+ was much higher for women with HPV16/18 infection (19.4%, 95% CI 15.8–23.8% with borderline/low‐grade cytology and 10.7%, 95% CI 8.3–13.9% with normal cytology) than for those with other HPV types (7.3%, 95% CI 5.4–9.7% with borderline/low‐grade cytology and 3.2%, 95% CI 2.2–4.5% with normal cytology). Among the 379 women with normal to low‐grade cytology and new HPV infection, the 10‐year cumulative CIN3+ risk was 2.9% (95% CI 1.6–5.2%).ConclusionsThe CIN3 risk is confined to women with persistent type‐specific HPV so partial genotyping test assays identifying HPV16/18 as a minimum are essential for efficient risk stratification. Immediate referral to colposcopy for HPV+ women with borderline or low‐grade cytology and referral after a year if still HPV+ with normal cytology may be unnecessary. Low‐grade lesions can safely be retested to identify those with persistent HPV. Recall intervals of 1 year for HPV16/18 and 2 years for other high‐risk HPVs are justified for women with normal cytology and might also be considered for women with borderline/low‐grade cytology. The minimal risk of invasive cancer that has progressed beyond stage 1A must be weighed against the advantages for patients and the NHS of reducing the number of referrals to colposcopy.Tweetable abstractCervical screening would be better for women and cheaper for the NHS if women with HPV and normal to low‐grade cytology were retested after a year or two when many infections will have cleared.