bluuebunny/crossref_metadata_2025 huggingface dataset:
The high persister phenotype ofPseudomonas aeruginosais associated with increased fitness and persistence in cystic fibrosis airways
Authors: Mojsoska Biljana , Cameron David R. , Bartell Jennifer A. , Haagensen Janus Anders Juul , Sommer Lea M. , Lewis Kim , Molin Søren , Johansen Helle Krogh
Published: February 2019

Despite intensive antibiotic treatment of cystic fibrosis (CF) patients,Pseudomonas aeruginosaoften persists in patient airways for decades, and can do so without the development of antibiotic resistance. Using a high-throughput screening assay of survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of high-persister variants (Hip) in a large patient cohort. In a screen of 467 longitudinal clinical isolates ofP. aeruginosafrom 40 CF patients, we classified 25.7% as Hip. Hip were identified in 26 patients, but only a few bacterial lineages were dominated by Hip. Instead, the emergence of Hip increased over time, suggesting that CF airways treated with ciprofloxacin select for Hip with an increased fitness in this environment. We generally observed diverse genetic changes in the Hip isolate population (as many co-occurring routes to increased fitness exist), but interestingly elevated mutation counts in the RpoN gene of 18 Hip isolates suggest that this sigma factor plays a role in shaping levels of antibiotic tolerance. To probe the impact of the Hip phenotype in a CF-similar environment, we tested the fitness properties of otherwise genotypically and phenotypically similar low-persister (Lop) and Hip isolates in co-culture using a specialized flow-cell biofilm system mimicking pharmacokinetic/-dynamic antibiotic dosing. Hip survived ciprofloxacin treatment far better than Lop isolates. The results of this investigation provide novel insights into persister dynamics and fitness contributions to survival in the CF lung, and show that the Hip phenotype of antibiotic susceptible bacteria plays an important role in long-term infections.

Significance

Antibiotic resistance is emphasized as a rapidly increasing health threat, but antibiotic tolerance via the occurrence of persister cells in antibiotic-treated bacterial populations is clinically and publicly neglected. In 40 CF patients representing a well-established human infection model – long-term lung infections byPseudomonas aeruginosa– we show the emergence and accumulation of persister variants in a clinical population heavily reliant on antibiotic therapy. We observe that the high-persister (Hip) phenotype is independent of resistance and likely the consequence of numerous genetic alterations, complicating surveillance and inhibition in the clinic. Furthermore, we find Hip are selected for over time, survive better than ‘normal’ bacteria, and can outcompete them in CF-similar conditions, ultimately affecting 65% of patients in an early disease cohort.
Protein kinase C is a key target for attenuation of Leigh syndrome by rapamycin
Authors: Martin-Perez Miguel , Ito Takashi K. , Grillo Anthony S. , Valente Anthony S. , Han Jeehae , Entwisle Samuel , Huang Heather Z. , Kim Dayae , Yajima Masanao , Kaeberlein Matt , Villén Judit
Published: February 2019

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. mTOR inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 KO mouse); however, the mechanism of rescue is unknown. Here we assessed the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice. We report that rapamycin remodels the brain proteome to alter mitochondrial structure, inhibits signaling through both mTOR complexes, and inhibits multiple protein kinase C (PKC) isoforms. Administration of PKC inhibitors was sufficient to increase survival, delay neurological deficits, and prevent hair loss in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.

One Sentence Summary

Proteomic and phosphoproteomic analysis of mouse brain identifies PKC as a key target to treat mitochondrial disease
Clinical significance of C4d deposition in renal tissues from patients with primary Sjögren’s syndrome—A preliminary study
Authors: Xia Wenli , Gao Bixia , Duan Lin , Li Yan , Wen Yubing , Chen Limeng , Li Xuemei , Zheng Falei , Li Mingxi
Published: February 2019

Objectives

To evaluate renal expression of C4d, a complement component in the classical/mannose binding lectin (MBL) pathway, in patients with primary Sjögren’s syndrome (pSS)-associated renal impairments.

Methods

We retrospectively reviewed the clinical and pathological data from 39 patients with pSS presenting with renal impairments. C4d was examined in paraffin-embedded biopsy tissues using immunohistochemistry. Glomerular C4d positive was defined when >75% glomeruli were globally stained. Tubulointerstitial C4d (TI-C4d) were scored semi-quantitatively as 0 (absent), 1 (spotty or weak), 2 (patchy) and 3 (diffuse). A TI-C4d score ≥2 was considered TI-C4d positive and included in the TI-C4d+ group and vice versa. Peritubular capillary (PTC) C4d was scored as 0 (absent), 1 (0∼10%, minimal), 2 (10%∼50%, focal), and 3 (>50%, diffuse).

Results

Glomerular C4d deposition was observed in all 8 patients with pSS-related membranous nephropathy (MN) without obvious C1q deposition. Two of 5 patients with mesangial proliferative glomerulonephritis and 1 of 2 patients with IgA nephropathy had mild mesangial C4d deposition. Sixteen patients (6 glomerular dominant and 10 tubulointerstitial dominant) presented TI-C4d score ≥2. Patients in the TI-C4d+ group exhibited a higher serum creatinine level at the time of renal biopsy (TI-C4d+ 132.5 [89.7, 165.5] vs. TI-C4d- 83.0 [70.7, 102.0] μmol/L, P=0.008). PTC C4d was observed in 12 patients, with each of minimal, focal and diffuse staining being noted in 4 patients.

Conclusions

The MBL pathway of complement activation was potentially involved in pSS-related MN. Tubulointerstitial C4d might be a pathological marker of severe renal injury in patients with pSS-related renal impairments.
Analyzing time-aggregated networks: the role of bootstrapping, permutation, and simulation
Authors: Bonnell T.R. , Vilette C.
Published: February 2019

Networks are often used to describe adaptive social systems, where individual (node) behaviour generates network-level structures that influence subsequent individual-level behaviour. To address questions about the dynamics of network structure in these systems, there is a need to analyze networks through time. Various statistical methods exist for estimating the behaviour of networks in time, in terms of both time-ordered and time-aggregated networks. In this paper, we discuss three main analytical steps for the analysis of time-aggregated network data: 1) aggregation choices, 2) null-model comparisons, and 3) constructing, parameterizing, and making inferences from time series models. We then present a custom R package, netTS, which facilitates these steps. Observed grooming data from a group of vervet monkeys, a highly social primate species, is used as an example to highlight three potential analyses: 1) quantifying the stability of network-level social structures through time, 2) identifying keystone nodes driving/maintaining network structures, and 3) quantifying the interdependence between node behaviour through time. In particular, we highlight the role of bootstrapping, permutation, and simulation as critical components in the analysis of time-aggregated networks.
Increased Neurite Orientation-Dispersion and Density in the TgCRND8 Mouse Model of Amyloidosis: Inverse Relation with Functional Connectome Clustering and Modulation by Interleukin-6
Authors: Colon-Perez Luis M. , Ibanez Kristen R. , Suarez Mallory , Torroella Kristin , Acuna Kelly , Ofori Edward , Levites Yona , Vaillancourt David E. , Golde Todd E. , Chakrabarty Paramita , Febo Marcelo
Published: February 2019

Extracellular β-amyloid (Aβ) plaque deposits and inflammatory immune activation are thought to alter various aspects of tissue microstructure, such as extracellular free water, fractional anisotropy and diffusivity, as well as the density and geometric arrangement of axonal processes. Quantifying these microstructural changes in Alzheimer’s disease and related neurodegenerative dementias could serve to accurately monitor or predict disease course. In the present study we used high-field diffusion magnetic resonance imaging (dMRI) to determine how Aβ and inflammatory interleukin-6 (IL6), alone or in combination, affectin vivotissue microstructure in the TgCRND8 mouse model of Alzheimer’s-type Aβ deposition. TgCRND8 and non-transgenic (nTg) mice expressing brain-targeted IL6 or enhanced glial fibrillary protein (EGFP controls) were scanned at 8 months of age using a 2-shell, 54-gradient direction dMRI sequence at 11.1 Tesla. Images were processed using the free water elimination method and the neurite orientation dispersion and density imaging (NODDI) model. DTI and NODDI processing in TgCRND8 mice revealed a microstructure pattern consistent with reduced white matter integrity along with an increase in density and geometric complexity of axonal and dendritic processes. This included reduced FA, mean diffusivity (MD), and free water (FW), and increased ‘neurite’ density (NDI) and orientation dispersion (ODI). IL6 produced a ‘protective-like’ effect on FA in TgCRND8 mice, although there were minimal microstructure changes in these mice compared IL6 expressing nTg mice. In addition, we found that NDI and ODI had an inverse relationship with the functional connectome clustering coefficient, which was affected by Aβ and IL6. The relationship between NODDI and graph theory metrics suggests that increasing the density and orientation dispersion of neurites may relate to diminished functional network organization in the brain.
Inverted translational control of eukaryotic gene expression by ribosome collisions
Authors: Park Heungwon , Subramaniam Arvind R.
Published: February 2019

The canonical model of eukaryotic translation posits that efficient translation initiation increases protein expression and mRNA stability. Contrary to this dogma, we show that increasing initiation rate can decrease both protein expression and stability of certain mRNAs in the budding yeast,S. cerevisiae. These mRNAs contain a stretch of poly-basic residues that cause ribosome stalling. Using computational modeling, we predict that the observed decrease in gene expression at high initiation rates occurs when ribosome collisions at stalls stimulate abortive termination of the leading ribosome and cause endonucleolytic mRNA cleavage. We test our prediction by identifying critical roles for the collision-associated quality control factors, Asc1 and Hel2 (RACK1 and ZNF598 in humans, respectively). Remarkably, hundreds ofS. cerevisiaemRNAs that contain ribosome-stall sequences also exhibit lower translation efficiency. We propose that these mRNAs have undergone evolutionary selection for inefficient initiation to escape collision-stimulated reduction in gene expression.
Modeling the Human Segmentation Clock with Pluripotent Stem Cells
Authors: Matsuda Mitsuhiro , Yamanaka Yoshihiro , Uemura Maya , Osawa Mitsujiro , Saito Megumu K. , Nagahashi Ayako , Nishio Megumi , Guo Long , Ikegawa Shiro , Sakurai Satoko , Kihara Shunsuke , Nakamura Michiko , Matsumoto Tomoko , Yoshitomi Hiroyuki , Ikeya Makoto , Yamamoto Takuya , Woltjen Knut , Ebisuya Miki , Toguchida Junya , Alev Cantas
Published: February 2019

Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation1. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types2,3, experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, and identified novel oscillatory genes in human iPSC-derived PSM. We furthermore determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial skeletal development such as spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like iPSC knock-out lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to recapitulate not only key features of human somitogenesis but also to provide novel insights into diseases associated with the formation and patterning of the human axial skeleton.
Stem cell derived human microglia transplanted in mouse brain to study genetic risk of Alzheimer’s Disease
Authors: Mancuso Renzo , Van Den Daele Johanna , Fattorelli Nicola , Wolfs Leen , Balusu Sriram , Burton Oliver , Sierksma Annerieke , Fourne Yannick , Poovathingal Suresh , Arranz-Mendiguren Amaia , Sala Frigerio Carlo , Claes Christel , Serneels Lutgarde , Theys Tom , Hugh Perry V. , Verfaillie Catherine , Fiers Mark , De Strooper Bart
Published: February 2019

Summary paragraph

Genetics highlight the central role of microglia in Alzheimer’s disease but at least 36% of AD-risk genes lack good mouse orthologues. Here, we show that embryonic stem cell (ESC)-derived human microglia successfully engraft the mouse brain and recapitulate transcriptionally primary human microglia derived from human surgical samples. Upon exposure to oligomeric Aβ a wide range of AD-risk genes are expressed that are not readily studied in current mouse models for AD. This work provides a unique humanized animal model that will allow elucidating the role of genetic risk in the pathogenesis of AD.
LD scores are associated with differences in allele frequencies between populations but LD score regression can still distinguish confounding from polygenicity
Authors: Alexander Mason , Curtis David
Published: February 2019

The LD score regression method tests whether there is an association between the LD score and allele frequency differences between cases and controls. It makes the assumption that there is no association between LD score and allele frequency differences between populations and hence that any observed association is due to a polygenic effect rather than population stratification. This assumption has not previously been tested. In comparisons between HapMap populations we observe that there is indeed an association between the LD score and allele frequency differences. However this effect is small and when we carry out simulations of large case-control samples the effect becomes negligible. We conclude that if the intercept is small then any increase in mean chi-squared does indeed reflect a polygenic effect rather than population stratification.
Field validation of an eDNA assay for the endangered white-clawed crayfishAustropotamobius pallipes
Authors: Atkinson Siobhán , Carlsson Jeanette E.L. , Ball Bernard , Kelly-Quinn Mary , Carlsson Jens
Published: February 2019

The white-clawed crayfishAustropotamobius pallipeshas undergone extensive declines within its native range in the last century. Because of its threatened status, European legislation requires the species to be regularly monitored and that Special Areas of Conservation (SACs) be designated for it. Knowledge on the distribution of this species is vital for addressing these needs. This study presents an environmental (e)DNA assay to detectA. pallipesin water samples, based on the mitochondrial cytochrome oxidase I (COI) gene, utilizing species-specific primers, a minor groove binding (MGB) probe and quantitative PCR. The results of this study indicate that eDNA is an effective tool for detectingA. pallipesin a lotic system, and could provide a valuable, non-invasive method for determining the distribution of this species.
Targeting endometrial cancer stem cell activity with metformin is inhibited by patient-derived adipocyte-secreted factors
Authors: Kitson Sarah J. , Rosser Matthew , Fischer Deborah P. , Marshall Kay M. , Clarke Robert B. , Crosbie Emma J.
Published: February 2019

Translational relevance

Endometrial cancer stem cells are reported to drive metastasis and disease relapse and are resistant to commonly used chemotherapeutic drugs. Here, we characterise ALDHhighand CD133+veendometrial cancer stem cells with differing levels of cancer stem cell activity and stem cell and epithelial-mesenchymal transition (EMT) gene expression. Their proportions and activity are reduced following treatment with metformin at a lower concentration than that required to affect bulk tumour cell proliferation, establishing a selective and specific effect on endometrial cancer stem cells. We demonstrate that this effect is abrogated when endometrial cancer cells are pre-treated with adipocyte conditioned media, indicating adipokine-mediated resistance to metformin. These results support future studies of metformin as an adjuvant therapy to reduce the risk of disease recurrence but should also investigate the impact of obesity on treatment response.

Purpose

Advanced endometrial cancer continues to have a poor prognosis due to limited treatment options, which may be further adversely impacted by obesity. Endometrial cancer stem cells have been reported to drive metastasis, chemotherapy resistance and disease relapse, but have yet to be fully characterised and no specific targeted therapies have been identified. Here, we describe the phenotype and genotype of aldehyde dehydrogenase high (ALDHhigh) and CD133+veendometrial cancer stem cells and how adipocyte secreted mediators block the inhibitory effect of metformin on endometrial cancer stem cell activity.

Experimental design

Ishikawa and Hec-1a cell lines were used to characterise ALDHhighand CD133+veendometrial cancer cells using flow cytometry, functional sphere assays and quantitative-Polymerase Chain Reaction. The comparative effect of metformin on endometrial cancer stem cell activity and bulk tumour cell proliferation was determined using an Aldefluor and cytotoxicity assay. The impact of adipocyte secreted mediators on metformin response was established using patient-derived conditioned media.

Results

ALDHhighcells demonstrated greater endometrial cancer stem cell activity than CD133+vecells and had increased expression of stem cell and epithelial-mesenchymal transition genes. Treatment with 0.5-1mM metformin reduced the proportion and activity of both endometrial cancer stem cell populations (p≤0.05), without affecting cell viability. This effect was, however, inhibited by exposure to patient-derived adipocyte conditioned media.

Conclusions

These results indicate a selective and specific effect of metformin on endometrial cancer stem cell activity, which is blocked by adipocyte secreted mediators. Future studies of metformin as an adjuvant therapy in endometrial cancer should be adequately powered to investigate the influence of body mass on treatment response.
Flowering behaviour inArabis alpinaensures the maintenance of a perennating dormant bud bank
Authors: Vayssières Alice , Mishra Priyanka , Roggen Adrian , Ponraj Udhaya , Neumann Ulla , Theres Klaus , Ljung Karin , Albani Maria C.
Published: February 2019

Arabis alpina, similar to woody perennials, has a complex architecture with a zone of axillary vegetative branches and a zone of dormant buds that serve as perennating organs. We show that floral development during vernalization is the key for shaping the dormant bud zone by facilitating a synchronized and rapid growth after vernalization and thereby causing an increase in auxin response and transport and endogenous indole-3-acetic acid (IAA) levels in the stem. Floral development during vernalization is associated with the development of axillary buds in subapical nodes. Our transcriptome analysis indicated that these buds are not dormant during vernalization but only attain sustained growth after the return to warm temperatures. Floral and subapical vegetative branches grow after vernalization and inhibit the development of the buds below. Dormancy in these buds is regulated across theA. alpinalife cycle by low temperatures and by apical dominance in a BRANCHED 1-dependent mechanism.
The effect of drought and intercropping on chicory nutrient uptake from below 2 m studied in a multiple tracer setup
Authors: Rasmussen Camilla Ruø , Thorup-Kristensen Kristian , Dresbøll Dorte Bodin
Published: February 2019

Aims

We tested if chicory acquires nutrients from soil layers down to 3.5 m depth and whether the deep nutrient uptake increases as a result of topsoil drought or topsoil resource competition. We also tested whether application of the trace elements Cs, Li, Rb, Sr, and Se, as tracers result in similar uptake rates.

Methods

The methodological tests were primarily carried out in a pilot experiment where the five tracers were applied to 1 m depth in lucerne and red beet grown in tube rhizotrons. The dynamics of deep nutrient uptake in chicory was studied in large 4 m deep rhizoboxes. A drought was imposed when roots had reached around 2 m depth.

Results

Chicory acquired tracers applied to 3.5 m depth, but we found no compensatory tracer uptake with depth during drought. We found some indications of a compensatory tracer uptake from 2.3 and 2.9 m depth in intercropped chicory. Application of equimolar amounts of trace elements resulted in similar excess tracer concentrations within species.

Conclusion

Chicory acquires nutrients from below 3 m but does not increase deep nutrient uptake as a response to limited topsoil nutrient availability.
The Ps and Qs of alarmone synthesis inStaphylococcus aureus
Authors: Yang Ning , Xie Shujie , Tang Nga-Yeung , Choi Mei Y. , Wang Ying , Watt Rory M.
Published: February 2019

During the stringent response, bacteria synthesize guanosine-3’,5’-bis(diphosphate) (ppGpp) and guanosine-5’-triphosphate 3’-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp ‘alarmones’ are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode ‘small alarmone synthetase’ (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5’-phosphate 3’-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins fromStaphylococcus aureus(Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. However, Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesisca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues fromEnterococcus faecalis(Ef-RelQ) andStreptococcus mutans(Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally-equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically stimulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp withinS. aureusand other Gram-positive bacterial species.
Can we predict which species win when new habitat becomes available?
Authors: Nomura Miki , Ohlemüller Ralf , Lee William G. , Lloyd Kelvin M. , Anderson Barbara J.
Published: February 2019

Land cover change is a key component of anthropogenic global environmental change, contributing to changes in environmental conditions of habitats. These changes can lead to the redistribution of species and shifts in the functional composition and properties of ecosystems. Deforestation is globally the most widespread anthropogenically driven land cover change leading to conversion from closed forest to open non-forest habitat. The consequences of these functional habitat changes on species distributions are only poorly understood. This study investigates the relative roles of geographic features, species climatic niche characteristics and species traits in determining the ability of open-habitat plant species to take advantage of recently opened habitats. We use current occurrence records of 18 herbaceous, predominantly open-habitat species of the genus Acaena (Rosaceae) to determine their prevalence in recently opened habitat. Geographic features of the spatial distribution of open habitat, species’ climatic niche characteristics, and species traits related to dispersal were tested their correlation with species’ prevalence in anthropogenically opened habitat. While primary open habitat (naturally open) was characterised by cold climates, secondary open habitat (naturally closed but anthropogenically opened) is characterised by warmer and wetter conditions. We found high levels of variation in the prevalence of secondary open habitat among the investigated species indicating differences between species in their ability to colonise newly opened habitat. For the species investigated, geographical and climatic niche factors showed generally stronger relationships with species’ prevalence in secondary open habitat than functional traits did. For small herbaceous species, geographical and environmental factors appear to be more important than species functional traits for facilitating expansion into secondary open habitats. Our results suggested that the land cover change might have triggered the shifts of factors controlling open-habitat plant distributions from the competition with forest trees to current environmental constraints.
Comparison of different orders of Legendre polynomials in random regression model for estimation of genetic parameters and breeding values of milk yield in the Chinese Holstein population
Authors: Li Jianbin , Gao Hongding , Madsen Per , Liu Wenhao , Bao Peng , Xue Guanghui , Yang Jun , Gao Yundong , Su Guosheng
Published: February 2019

Random regression test-day model has become the most commonly adopted model for routine genetic evaluations for different dairy populations, which allows accurately accounting for genetic and environmental effects at different periods during lactation. The objective of this study was to explore appropriate random regression test-day model for genetic evaluation of milk yield in Chinese Holstein population. Data included 419,567 test-day records from 54,417 cows in the first lactation. Variance components and breeding values were estimated using random regression test-day model with different order (first order to fifth order) of Legendre polynomials, and accounted for homogeneous or heterogeneous residual variance across the lactation. The goodness of fit of the models was evaluated by total residual variance (TRV) and − 2logL. Further, the predictive ability of the models was assessed by Spearman’s rank correlation between estimated breeding values for 305d milk yield (EBV305) from the full data set and reduced data set in which the records from the last calving year were masked. The results showed that random regression models using third order Legendre polynomials (LP3) with heterogeneous residual variance achieved the lower TRV and − 2logL value and the highest correlation for EBV305 between full data and reduced data. Heritability estimated by this model was 0.250 for 305d milk yield and ranged from 0.163 to 0.304 for test-day milk yield. We suggest random regression model with Legendre polynomial of order 3 and accounting for heterogeneous residual variances could be an appropriate model to be used for genetic evaluation of milk yield for Chinese Holstein population.
Photoperiod-induced neurotransmitter plasticity declines with aging: an epigenetic regulation?
Authors: Pritchard Rory , Chen Helene , Romoli Ben , Spitzer Nicholas C. , Dulcis Davide
Published: February 2019

Neuroplasticity has classically been understood to arise through changes in synaptic strength or synaptic connectivity. A newly discovered form of neuroplasticity, neurotransmitter switching, involves changes in neurotransmitter identity. Chronic exposure to different photoperiods alters the number of dopamine (tyrosine hydroxylase, TH+) and somatostatin (SST+) neurons in the paraventricular nucleus (PaVN) of the hypothalamus of adult rats and results in discrete behavioral changes. Here we investigate whether photoperiod-induced neurotransmitter switching persists during aging and whether epigenetic mechanisms of histone acetylation and DNA methylation may contribute to this neurotransmitter plasticity. We show that this plasticity is robust at 1 and at 3 months but reduced in TH+ neurons at 12 months and completely abolished in both TH+ and SST+ neurons by 18 months. De novo methylation and histone 3 acetylation were observed following short-day photoperiod exposure in both TH+ and SST+ neurons at 1 and 3 months while an overall increase in methylation of SST+ neurons paralleled neuroplasticity reduction at 12 and 18 months. Histone acetylation increased in TH+ neurons and decreased in SST+ neurons following short-day exposure at 3 months while the total number of acetylated PaVN neurons remained constant. Reciprocal histone acetylation in TH+ and SST+ neurons suggests the importance of studying epigenetic regulation at the circuit level for identified cell phenotypes. The association of age-dependent reduction in neurotransmitter plasticity and changes in DNA methylation and acetylation patterns in two neuronal phenotypes known to switch transmitter identity suggests mechanistic insights into transmitter plasticity in the aging brain.

SIGNIFICANCE

Neurotransmitter switching, like changes in synaptic strength, formation of new synapses and synapse remodeling, declines with age. This age-dependent reduction in transmitter plasticity is associated with changes in levels of DNA methylase and histone deacetylase that imply epigenetic regulation of transcription. A reciprocal pattern of histone acetylation in a single population of neurons that depends on the transmitter expressed emphasizes the value of studying epigenetic mechanisms at the level of cell phenotypes rather than cell genotypes or whole tissue. The findings may be useful for developing approaches for non-invasive treatment of disorders characterized by neurotransmitter dysfunction.
Integrated Phosphoproteomics and Transcriptional Classifiers Reveal Hidden RAS Signaling Dynamics in Multiple Myeloma
Authors: Lin Yu-Hsiu T. , Way Gregory P. , Barwick Benjamin G. , Mariano Margarette C. , Marcoulis Makeba , Ferguson Ian D. , Driessen Christoph , Boise Lawrence H. , Greene Casey S. , Wiita Arun P.
Published: February 2019

A major driver of multiple myeloma is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employ an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in multiple myeloma to inform precision medicine strategies. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. Transcriptional analysis suggests that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs. WT RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes whereas RAS mutations overall carry no survival impact. We further develop an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. These results may lead to improved stratification of MM patients in clinical trials while also revealing unexplored modes of Ras biology.
Altered Cell-Substrate Behavior on Microporous Membranes is a Result of Disruption and Grip
Authors: Allahyari Zahra , Gholizadeh Shayan , Chung Henry H. , Delgadillo Luis F. , Gaborski Thomas R.
Published: February 2019

Porous membranes are ubiquitous in cell co-culture and tissue-on-a-chip studies. These materials are predominantly chosen for their semi-permeable and size exclusion properties to restrict or permit transmigration and cell-cell communication. However, previous studies have shown pore size, spacing and orientation affect cell behavior including extracellular matrix production and migration. The mechanism behind this behavior is not fully understood. In this study, we fabricated micropatterned non-fouling polyethylene glycol (PEG) islands to mimic pores in order to decouple the effect of surface discontinuity from grip provided by pore wall edges. Similar to porous membranes, we found that the PEG islands hindered fibronectin fibrillogenesis with cells on patterned substrates producing shorter fibrils. Additionally, cell migration speed over micropatterned PEG islands was greater than unpatterned controls, suggesting that disruption of cell-substrate interactions by PEG islands promoted a more dynamic and migratory behavior, similarly to cells migrating on microporous membranes. Preferred cellular directionality during migration was nearly identical between substrates with identically patterned PEG islands and micropores, further confirming disruption of cell-substrate interactions as a common mechanism behind the cellular responses on these substrates. Interestingly, cell spreading and the magnitude of migration speed was significantly greater on porous membranes compared to PEG islands with identical feature size and spacing, suggesting pore edges enhanced cellular grip. These results provide a more complete picture on how porous membranes affect cells which are grown on them in an increasing number of cellular barrier and co-culture studies.
Dynamic chromatin targeting of BRD4 stimulates cardiac fibroblast activation
Authors: Stratton Matthew S. , Bagchi Rushita A. , Hirsch Rachel A. , Riching Andrew S. , Felisbino Marina B. , Enyart Blake Y. , Koch Keith A. , Cavasin Maria A. , Alexanian Michael , Song Kunhua , Qi Jun , Lemieux Madeleine E. , Lam Maggie P.Y. , Haldar Saptarsi M. , Lin Charles Y. , McKinsey Timothy A.
Published: February 2019

Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in pre-clinical models of heart failure (HF). However, the mechanisms by which BRD4 promotes pathological myocardial fibrosis remain unclear. Here, we demonstrate that BRD4 functions as an effector of TGF-β signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the SERTA domain-containing protein 4 (Sertad4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 mitogen-activated protein kinase, and provide evidence of a novel function for Sertad4 in TGF-β-mediated cardiac fibroblast activation. These findings define BRD4 as a central regulator of the pro-fibrotic cell state of cardiac fibroblasts, and establish a signaling circuit for epigenetic reprogramming in HF.
An E460D substitution in the NS5 protein of tick-borne encephalitis virus confers resistance to the inhibitor Galidesivir (BCX4430) and also attenuates the virus for mice
Authors: Eyer Ludek , Nougairède Antoine , Uhlířová Marie , Driouich Jean-Sélim , Zouharová Darina , Valdés James J. , Haviernik Jan , Gould Ernest A. , De Clercq Erik , de Lamballerie Xavier , Ruzek Daniel
Published: February 2019

The adenosine analogue Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a Phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola virus infection. Moreover, Galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution, in the active site of TBEV RNA-dependent-RNA-polymerase (RdRp), confers resistance to Galidesivir in cell culture. Stochastic molecular simulations indicate that the steric freedom caused by the E460D substitution increases close electrostatic interactions between the inhibitor and the interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an incorrect/modified nucleotide. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2’-C-methyladenosine, 2’-C-methyladenosine and 4’-azido-aracytidine. Although, the E460D substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors and the potential contribution of viral RdRp to flavivirus neurovirulence.

Importance

Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. We have previously found that Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola virus infection, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to Galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of viral RNA genome. Although, this substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity.
UDCT: Unsupervised data to content transformation with histogram-matching cycle-consistent generative adversarial networks
Authors: Ihle Stephan , Reichmuth Andreas M. , Girardin Sophie , Han Hana , Stauffer Flurin , Bonnin Anne , Stampanoni Marco , Vörös János , Forró Csaba
Published: February 2019

The segmentation of images is a common task in a broad range of research fields. To tackle increasingly complex images, artificial intelligence (AI) based approaches have emerged to overcome the shortcomings of traditional feature detection methods. Owing to the fact that most AI research is made publicly accessible and programming the required algorithms is now possible in many popular languages, the use of such approaches is becoming widespread. However, these methods often require data labeled by the researcher to provide a training target for the algorithms to converge to the desired result. This labeling is a limiting factor in many cases and can become prohibitively time consuming. Inspired by Cycle-consistent Generative Adversarial Networks’ (cycleGAN) ability to perform style transfer, we outline a method whereby a computer generated set of images is used to segment the true images. We benchmark our unsupervised approach against a state-of-the-art supervised cell-counting network on the VGG Cells dataset and show that it is not only competitive but can also precisely locate individual cells. We demonstrate the power of this method by segmenting bright-field images of cell cultures, a live-dead assay of C.Elegans and X-ray-computed tomography of metallic nanowire meshes.
Dinucleoside polyphosphates act as 5’-RNA caps inEscherichia coli
Authors: Hudeček Oldřich , Benoni Roberto , Culka Martin , Hubálek Martin , Rulíšek Lubomír , Cvačka Josef , Cahová Hana
Published: February 2019

Dinucleoside polyphosphates (NpnNs), discovered more than 50 years ago,1are pleiotropic molecules present in almost all types of cells.2It has been shown that their intracellular concentration can under stress conditions increase from the µM to mM range2,3. However, the cellular roles and mechanisms of action of NpnNs are still speculative4,5. They have never been considered as part of the RNA, even though they have similar chemical structures as already known RNA caps, such as the nicotinamide adenine dinucleotide (NAD)6-8and 7-methylguanylate cap9. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps inEscherichia coli(E. coli). NpnNs are excellent substrates for T7 andE. coliRNA polymerases (RNAP) and efficiently initiate transcription. Further, we demonstrate that theE. colidecapping enzyme RNA 5’ pyrophosphohydrolase (RppH) is able to remove the NpnNs-cap from the RNA. RppH was, however, not able to cleave the methylated forms of the NpnN-caps, suggesting that the methylation adds an additional layer to the RNA stability regulation. Our work introduces an original perspective on the chemical structure of RNA in prokaryotes and the function of RNA caps. This is the first evidence that small molecules like NpnNs can act in cells via their incorporation into RNA and influence the cellular metabolism.
Erythropoiesis differences in various clinical phases of Dengue fever using Immature Reticulocyte Fraction parameter
Authors: Oehadian Amaylia , Vidyaniati Putri , Candra Jeffery Malachi , Sumardi Uun , Susandi Evan , Alisjahbana Bachti
Published: February 2019

Dengue fever is the most common cause of hospitalization in otherwise healthy person. The incidence rate increases significantly every year, from 0.05 per 100.000 inhabitants in 1968 to 35-40 per 100.000 inhabitants in 2013. Thrombocytopenia and leucopenia are the most commonly reported changes, whether they are caused by bone marrow suppression or peripheral destruction. Anemia has also been observed to occur in dengue infection with unknown mechanism. Immature Reticulocyte Fraction (IRF) is a parameter reflecting the most immature reticulocyte fraction and it can identify the earliest stage of erythropoiesis disorder. This research aimed to determine the mechanism of erythropoiesis disorders that led to anemia using IRF parameter in the various clinical phases of dengue fever. This study was a comparative analytical research using secondary data derived from theDengue-associated Endothelial Cell Dysfunction and Thrombocyte Activation(DECENT) research. The baseline characteristic data consists of sex, age, level of hemoglobin, hematocrit, leucocyte, and thrombocyte, also the IRF. The patients were grouped into the fever, critical, recovery, and convalescent phases, plus healthy control group. The data was analyzed using the Kolmogorov-Smirnov normality test, followed by Friedman test and Mann-Whitney post hoc test. There were 244 research subjects and the median age was 24 (14-67), with similar ratio of male and female. The median IRF for all the research subjects was 4.8% with an IQR of 2.4-8.1%. The fever-phase group showed a median of 1.8% with an IQR of 0.5-2.85%. The critical-phase group showed a median of 3.6% with an IQR of 1.8- 5.0%, while the median for the recovery-phase group was 7.05% with an IQR of 4.08-11.85%. The convalescent-phase group showed a median of 7.3 % with an IQR of 3.95-9.3%, and the healthy-control group showed a median of 4.1% with an IQR of 2.2-6.6%. There was a significant difference in IRF between the groups (p<0.05). Immature Reticulocyte Fraction in fever phase was significantly different with IRF in other phases and healthy controls (p<0.05). In the critical phase, IRF was not significantly different from the healthy controls (p=0.218). Summarizing, there are changes in erythropoiesis activity in various clinical phases of dengue infection. Erythropoiesis suppression occurred mainly during the fever phase and it started to restore in the critical phase. In the recovery and convalescent phases, the erythropoiesis activity was increased.
Extension of the transferable aspherical pseudoatom data bank for the comparison of molecular electrostatic potentials in structure–activity studies
Authors: Kumar Prashant , Gruza Barbara , Bojarowski Sławomir Antoni , Dominiak Paulina Maria
Published: February 2019

The transferable aspherical pseudoatom data bank, UBDB2018, is extended with over 130 new atom types present in small and biological molecules of great importance in biology and chemistry. UBDB2018 can be applied either as a source of aspherical atomic scattering factors in a standard X-ray experiment (d
min ≃ 0.8 Å) instead of the independent atom model (IAM), and can therefore enhance the final crystal structure geometry and refinement parameters; or as a tool to reconstruct the molecular charge-density distribution and derive the electrostatic properties of chemical systems for which 3D structural data are available. The extended data bank has been extensively tested, with the focus being on the accuracy of the molecular electrostatic potential computed for important drug-like molecules, namely the HIV-1 protease inhibitors. The UBDB allows the reconstruction of the reference B3LYP/6-31G** potentials, with a root-mean-squared error of 0.015 e bohr−1 computed for entire potential grids which span values from ca 200 e bohr−1 to ca −0.1 e bohr−1 and encompass both the inside and outside regions of a molecule. UBDB2018 is shown to be applicable to enhancing the physical meaning of the molecular electrostatic potential descriptors used to construct predictive quantitative structure–activity relationship/quantitative structure–property relationship (QSAR/QSPR) models for drug discovery studies. In addition, it is suggested that electron structure factors computed from UBDB2018 may significantly improve the interpretation of electrostatic potential maps measured experimentally by means of electron diffraction or single-particle cryo-EM methods.
Shape transform phasing of edgy nanocrystals
Authors: Chen J. P. J. , Donatelli J. J. , Schmidt K. E. , Kirian R. A.
Published: February 2019

Diffraction patterns from small protein crystals illuminated by highly coherent X-rays often contain measurable interference signals between Bragg peaks. This coherent `shape transform' signal introduces enough additional information to allow the molecular densities to be determined from the diffracted intensities directly, without prior information or resolution restrictions. However, the various correlations amongst molecular occupancies/vacancies at the crystal surface result in a subtle yet critical problem in shape transform phasing whereby the sublattices of symmetry-related molecules exhibit a form of partial coherence amongst lattice sites when an average is taken over many crystal patterns. Here an iterative phase retrieval algorithm is developed which is capable of treating this problem; it is demonstrated on simulated data.