bluuebunny/crossref_metadata_2025 huggingface dataset:
Ventilator-associated events, not ventilator-associated pneumonia, is associated with higher mortality in trauma patients
Authors: Meagher Ashley D. , Lind Margaret , Senekjian Lara , Iwuchukwu Chinenye , Lynch John B. , Cuschieri Joseph , Robinson Bryce R.H.
Published: April 2019

BACKGROUND

Ventilator-associated events (VAE), using objective diagnostic criteria, are the preferred quality indicator for patients requiring mechanical ventilation (MV) for greater than 48 hours. We aim to identify the occurrence of VAE in our trauma population, the impact on survival, and length of stay, as compared to the traditional definition of ventilator-associated pneumonia (VAP).

METHODS

This retrospective review included adult trauma patients, who were Washington residents, admitted between 2012 and 2017, and required at least 3 days of MV. Exclusions included patients with Abbreviated Injury Scale head score greater than 4 and burn related mechanisms of injury. We matched trauma registry data with our institutional, physician-adjudicated, and culture-confirmed ventilator event database. We compared the clinical outcomes of ventilator-free days, intensive care unit length of stay, hospital length of stay, and likelihood of death between VAE and VAP.

RESULTS

One thousand five hundred thirty-three trauma patients met criteria; 124 (8.1%) patients developed VAE, 114 (7.4%) patients developed VAP, and 63 (4.1%) patients met criteria for both VAE and VAP. After adjusted analyses, patients with VAE were more likely to die (hazard ratio [HR], 2.86; 95% confidence interval [CI], 1.44–5.68), than those with VAP, as well those patients with neither diagnosis (HR, 2.83; 95% CI, 1.83–4.38). Patients with VAP were no more likely to die (HR, 1.55; 95% CI, 0.91–2.68) than those with neither diagnosis. Patients with VAE had fewer ventilator-free days than those with VAP (HR, −2.71; 95% CI, −4.74 to −0.68).

CONCLUSION

Critically injured trauma patients who develop VAE are three times more likely to die and utilize almost 3 days more MV than those that develop VAP. The objective criteria of VAE make it a promising indicator on which quality indicator efforts should be focused. Future studies should be aimed at identification of modifiable risk factors for VAE and their impact on outcome, as these patients are at high risk for death.

LEVEL OF EVIDENCE

Retrospective cohort study, level III.
Cardiorespiratory fitness predicts greater hippocampal volume and rate of episodic associative learning in older adults
Authors: Cole Rachel C. , Hazeltine Eliot , Weng Timothy B. , Wharff Conner , DuBose Lyndsey E. , Schmid Phillip , Sigurdsson Gardar , Magnotta Vincent A. , Pierce Gary L. , Voss Michelle W.
Published: April 2019

Declining episodic memory is common among otherwise healthy older adults, in part due to negative effects of aging on hippocampal circuits. However, there is significant variability between individuals in severity of aging effects on the hippocampus and subsequent memory decline. Importantly, variability may be influenced by modifiable protective physiological factors such as cardiorespiratory fitness (CRF). More research is needed to better understand which aspects of cognition that decline with aging benefit most from CRF. The current study evaluated the relation of CRF with learning rate in the Episodic Associative Learning (EAL) task, a task designed specifically to target hippocampal-dependent relational binding and to evaluate learning with repeated occurrences. Results show that higher CRF was associated with larger hippocampal volume and faster learning rate. Larger hippocampal volume was also associated with faster learning rate, and hippocampal volume partially mediated the relationship between CRF and learning rate. Further, to support the distinction between learning item relations and learning higher-order sequences, which declines with aging but is largely reliant on extra-hippocampal learning systems, we found that EAL learning rate was not related to motor sequence learning on the alternating serial reaction time task. Motor sequence learning was also not correlated with hippocampal volume. Thus, for the first time we show that higher CRF in healthy older adults is related to enhanced rate of relational memory acquisition, in part mediated by benefits on the hippocampus.
Deficient stereopsis in the normal population revisited: why current clinical stereo tests may not be adequate
Authors: Hess Robert F , Dillon Rebecca , Ding Rifeng , Zhou Jiawei
Published: April 2019

Significance statement

Applied applications for occupational screening, clinical tests should assess sensitivity to the sign as well as the magnitude of disparity.

Purpose

To determine why the high incidence of stereo anomaly found using laboratory tests with polarity-based increment judgements (i.e., depth sign) is not reflected in clinical measurements that involve single-polarity incremental judgements (i.e., depth magnitude).

Methods

An iPod-based measurement that involved the detection of an oriented shape defined by a single polarity-depth increment within a random dot display was used. A staircase procedure was used to gather sufficient trials to derive a meaningful measure of variance for the measurement of stereopsis over a large disparity range. Forty-five adults with normal binocular vision (20 - 65 years old) and normal or corrected-to-normal (0 logMAR or better) monocular vision participated in this study.

Results

Observers’ stereo acuities ranged between 10 and 100 arc seconds, and were normally distributed on a log scale (p = 0.90, 2-tailed Shapiro-Wilk test). The present results using a single polarity depth increment task (i.e., depth magnitude) show a similar distribution to those using a similar task using the Randot preschool stereo test on individuals between the ages of 19-35 using either the 4-book test (n = 33) or the 3-book test (n = 40), but very different results when the iPod test involved a polarity-based increment judgement (i.e., depth sign).

Conclusions

The present clinical stereo tests are based on magnitude judgements and are unable to detect the high percentage of stereo anomalous individuals in the normal population revealed using depth sign judgements.
CRISPR-Cas9 Gene Editing in Lizards Through Microinjection of Unfertilized Oocytes
Authors: Rasys Ashley M. , Park Sungdae , Ball Rebecca E. , Alcala Aaron J. , Lauderdale James D. , Menke Douglas B.
Published: April 2019

CRISPR-Cas9 mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are currently no reptiles with effective methods for targeted mutagenesis. Here we present a new approach that enables the efficient production of CRISPR-Cas9 induced mutations in Anolis lizards, an important model for studies of reptile evolution and development.
The mutational landscape of a prion-like domain
Authors: Bolognesi Benedetta , Faure Andre J. , Seuma Mireia , Schmiedel Jörn M. , Tartaglia Gian Gaetano , Lehner Ben
Published: April 2019

Specific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases1–5. For example, cytoplasmic aggregates of the RNA-binding protein TDP-43 are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS)6,7. However, it is still unclear for ALS and other diseases whether it is the insoluble aggregates or other forms of the mutated proteins that cause these diseases that are actually toxic to cells8–13. Here we address this question for TDP-43 by systematically mutating14the protein and quantifying the effects on cellular toxicity. We generated >50,000 mutations in the intrinsically disordered prion-like domain (PRD) and observed that changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation actually reduce cellular toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquid-like condensates. The genetic interactions in double mutants reveal that specific structures exist in this ‘unstructured’ regionin vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins as well as theirin vivostructural conformations. Moreover, we show that aggregation of TDP-43 is not harmful but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase.
Microsaccades and attention in a high-acuity visual alignment task
Authors: Nanjappa Rakesh , McPeek Robert M.
Published: April 2019

While aiming and shooting, we make tiny eye movements called microsaccades that shift gaze between task-relevant objects within a small region. However, in the brief period before pressing trigger, microsaccades are suppressed. This might be due to the lack of the requirement to shift gaze as the retinal images of the two objects start overlapping on fovea. Or we might be actively suppressing microsaccades to prevent any disturbances in visual perception caused by microsaccades around the time of their occurrence and their subsequent effect on shooting performance.

In this study we looked at microsaccade rate while participants performed a simulated shooting task under two conditions: normal viewing in which they moved their eyes freely and eccentric condition in which they maintained gaze on a fixed target while performing shooting task at 5° eccentricity. As expected, microsaccade rate dropped at the end of the task in the normal viewing condition. However, we found the same for the eccentric condition in which microsaccade did not shift gaze between the task objects.

Microsaccades are also produced in response to shifts in covert attention. To test whether disengagement of covert attention from eccentric shooting location caused the drop in microsaccade rate, we monitored participant’s spatial attention location by employing a RSVP task simultaneously at a location opposite to the shooting task. Target letter detection at RSVP location did not improve during the drop in microsaccade rate, suggesting that covert attention was maintained at the shooting task location.

We conclude that in addition to their usual gaze-shifting function, microsaccades during fine acuity tasks might be modulated by cognitive processes other than spatial attention.
Heritability of skewed X-inactivation in female twins is tissue-specific and dependent on age
Authors: Zito Antonino , Davies Matthew N. , Tsai Pei-Chien , Roberts Susanna , Nardone Stefano , Bell Jordana T. , Wong Chloe C. Y. , Small Kerrin S.
Published: April 2019

To balance the X-linked transcriptional dosages between the sexes, one of the two X-chromosomes is randomly selected to be inactivated in somatic tissues of female placental mammals. Non-random, or skewed X-chromosome inactivation (XCI) toward one parental X has been observed in female somatic tissues, and this skewing effect has been associated with several complex human traits. However, the extent of the influence of genetic and environmental factors on XCI skewing is largely unknown. Here, we use RNA-seq and DNA-seq data taken from a large cohort of female twins to quantify the degree of skewing of XCI (DS) in multiple tissues and to study the relationship of XCI with age, genetic factors and complex traits. We show that the XCI patterns are highly tissue-specific with a higher prevalence of skewed XCI in blood-derived tissues than in fat or skin tissue. We also show that the DS in blood-derived tissues is associated with age and that the acquired DS occurs uniquely in blood-derived tissues with an inflection point at approximately 55 years of age. Heritability analysis indicates that the heritability of DS is both age and tissue specific; DS is heritable in blood tissue of females >55 years-old (h2= 0.34) but is not heritable in blood tissues of females <55 years-old (h2= 0), nor in skin and fat tissues at any age. We find a positive association between the DS and smoking status in blood tissues of older females (P= 0.02). The high tissue specificity of XCI patterns in human indicates the existence of tissue-specific mechanisms influencing XCI patterns, including genetic and environmental factors. We conclude that the heritability of XCI skewing in blood-derived tissues is dependent on age, representing a Gene x Age interaction that can shift the functional allelic dosage of an entire chromosome in a tissue-restricted manner.
Neurocognitive reorganization between crystallized intelligence, fluid intelligence and white matter microstructure in two age-heterogeneous developmental cohorts
Authors: Simpson-Kent Ivan L. , Fuhrmann Delia , Bathelt Joe , Achterberg Jascha , Borgeest Gesa Sophia ,   the CALM Team, Kievit Rogier A.
Published: April 2019

Despite the reliability of intelligence measures in predicting important life outcomes such as educational achievement and mortality, the exact configuration and neural correlates of cognitive abilities remain poorly understood, especially in childhood and adolescence. Therefore, we sought to elucidate the factorial structure and neural substrates of child and adolescent intelligence using two cross-sectional, developmental samples (CALM: N=551 (N=165 imaging), age range: 5-18 years, NKI-Rockland: N=337 (N=65 imaging), age range: 6-18 years). In a preregistered analysis, we used structural equation modelling (SEM) to examine the neurocognitive architecture of individual differences in childhood and adolescent cognitive ability. In both samples, we found that cognitive ability in lower and typical-ability cohorts is best understood as two separable constructs, crystallized and fluid intelligence, which became more distinct across development, in line with the age differentiation hypothesis. Further analyses revealed that white matter microstructure, most prominently the superior longitudinal fasciculus, was strongly associated with crystallized (gc) and fluid (gf) abilities. Finally, we used SEM trees to demonstrate evidence for developmental reorganization of gc and gf and their white matter substrates such that the relationships among these factors dropped between 7-8 years before increasing around age 10. Together, our results suggest that shortly before puberty marks a pivotal phase of change in the neurocognitive architecture of intelligence.
AlbaTraDIS: Comparative analysis of large datasets from parallel transposon mutagenesis experiments
Authors: Page Andrew J. , Bastkowski Sarah , Yasir Muhammad , Turner A. Keith , Le Viet Thanh , Savva George M. , Webber Mark A. , Charles Ian G.
Published: April 2019

Background

Bacteria have evolved over billions of years to survive in a wide range of environments. Currently, there is an incomplete understanding of the genetic basis for mechanisms underpinning survival in stressful conditions, such as the presence of anti-microbials. Transposon mutagenesis has been proven to be a powerful tool to identify genes and networks which are involved in survival and fitness under a given condition by simultaneously assaying the fitness of millions of mutants, thereby relating genotype to phenotype and contributing to an understanding of bacterial cell biology. A recent refinement of this approach allows the roles of essential genes in conditional stress survival to be inferred by altering their expression. These advancements combined with the rapidly falling costs of sequencing now allows comparisons between multiple experiments to identify commonalities in stress responses to different conditions. This capacity however poses a new challenge for analysis of multiple data sets in conjunction.

Results

To address this analysis need, we have developed ‘AlbaTraDIS’; a software application for rapid large-scale comparative analysis of TraDIS experiments that predicts the impact of transposon insertions on nearby genes. AlbaTraDIS can identify genes which are up or down regulated, or inactivated, between multiple conditions, producing a filtered list of genes for further experimental validation as well as several accompanying data visualisations. We demonstrate the utility of our new approach by applying it to identify genes used byEscherichia colito survive in a wide range of different concentrations of the biocide Triclosan. AlbaTraDIS automatically identified all well characterised Triclosan resistance genes, including the primary target,fabI. A number of new loci were also implicated in Triclosan resistance and the predicted phenotypes for a selection of these were validated experimentally and results showed high consistency with predictions.

Conclusions

AlbaTraDIS provides a simple and rapid method to analyse multiple transposon mutagenesis data sets allowing this technology to be used at large scale. To our knowledge this is the only tool currently available that can perform these tasks. AlbaTraDIS is written in Python 3 and is available under the open source licence GNU GPL 3 fromhttps://github.com/quadram-institute-bioscience/albatradis.
In vitro Evaluation of Antifungal Drug Combinations against Multidrug-resistant Candida auris isolates from New York Outbreak
Authors: O’Brien Brittany , Chaturvedi Sudha , Chaturvedi Vishnu
Published: April 2019

Since 2016, New York hospitals and healthcare facilities have faced an unprecedented outbreak of pathogenic yeast Candida auris. We tested over one thousand C. auris isolates from affected facilities and found high-resistance to fluconazole (FLC, MIC50>256 mg/L), and variable resistance to other antifungal drugs. Therefore, we evaluated if two-drug combinations are effective in vitro against multidrug-resistant C. auris. Broth micro-dilution (BMD) plates were custom-designed, and quality controlled by TREK Diagnostic System. We used MIC100 endpoints for the drug combination readings as reported earlier for the intra- and inter-laboratory agreements against Candida species and Aspergillus fumigatus. The study results were derived from 12,960 MIC100 readings, for fifteen C. auris isolates tested against 864 possible two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/L potentiated the most successful combinations with other drugs. Micafungin (MFG), Anidulafungin (AFG), Caspofungin (CAS) at individual concentrations of 0.25 mg/L in combination with 5FC (1.0 mg/L) yielded MIC100 for 14, 13, and 12 of 15 C. auris test isolates. AMB / 5FC (0.25/1.0 mg/L) yielded MIC100 for 13 isolates. None of the combinations were effective for C. auris 18-1, which tested resistant against FLC and 5FC, except POS/5FC (0.12/1.0 mg/L). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to deal with multidrug-resistant C. auris.
Complexity measures of the electroencephalograph capture loss and recovery of consciousness in patients anesthetized with propofol
Authors: Eagleman Sarah L. , Chander Divya , Reynolds Christina , Ouellette Nicholas T. , MacIver M. Bruce
Published: April 2019

Propofol is one of the most widely used anesthetics for routine surgical anesthesia. Propofol administration alone produces EEG spectral characteristics similar to most hypnotics; however, inter-individual variation can make spectral measures inconsistent. Complexity measures of EEG signals could offer universal measures to better capture anesthetic depth as brain activity exhibits nonlinear behavior at several scales. We tested the potential of nonlinear dynamics analyses to identify loss and recovery of consciousness at clinically relevant timepoints. Patients undergoing propofol general anesthesia for various surgical procedures were identified as having changes in states of consciousness by the loss and recovery of response to verbal stimuli after induction and upon cessation of anesthesia, respectively. Nonlinear dynamics analyses showed more significant differences between consciousness states than most spectral measures. Thus, complexity measures could provide a means for reliably capturing depth of consciousness based on subtle EEG changes at the beginning and end of anesthesia administration.
Attentive red squirrel mothers have faster-growing pups and higher lifetime reproductive success
Authors: Westrick Sarah E , Taylor Ryan W , Boutin Stan , Lane Jeffrey E , McAdam Andrew G , Dantzer Ben
Published: April 2019

Parental investment theory predicts that observed levels of parental care afforded to offspring are set by the benefits (to offspring quality and survival) relative to the costs (to parental survival or future reproduction). Although difficult to document in mammals, there is often substantial individual-variation in the amount of parental care within species. We measured the impact of individual variation in maternal care (“attentiveness” towards offspring or maternal motivation) on offspring growth and survival in a wild population of North American red squirrels (Tamiasciurus hudsonicus). We used latency to return to pups following a nest intrusion as a measure of maternal attentiveness to pups. We found this behavior to be repeatable within individuals suggesting this behavior is a personality trait or a “maternal style”. In this population, postnatal growth rate is important for pup overwinter survival. Pups from large litters grew faster if they had a highly attentive mother, indicating that maternal care behavior can mitigate the trade-off between litter size and offspring growth and potentially improve survival of pups. Additionally, more attentive mothers had slightly higher lifetime reproductive success than less attentive mothers. These results highlight important fitness effects of having a highly attentive mother and show that maternal care behavior can alter a fundamental life history trade-off between offspring quantity and quality.

Lay Summary

It pays to be attentive to your pups as a squirrel mom. In a long-term study of a wild population of North American red squirrels, we observed repeatable individual variation in maternal attentiveness towards offspring. Mothers who returned faster to pups following a nest intrusion produced faster growing pups and were able to produce larger fast-growing litters. Over their entire lifetime, attentive mothers also had more offspring recruit into the breeding population.
Methylomic profiles reveal sex-specific shifts in leukocyte composition associated with post-traumatic stress disorder
Authors: Kim Grace S. , Smith Alicia , Xu Fei , Michopoulos Vasiliki , Lori Adriana , Armstrong Don L. , Aiello Allison E. , Koenen Karestan C. , Galea Sandro , Wildman Derek E. , Uddin Monica
Published: April 2019

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder precipitated by trauma exposure. However, only some persons exposed to trauma develop PTSD. There are sex differences in risk; twice as many women as men, develop a lifetime diagnosis of PTSD. Methylomic profiles derived from peripheral blood are well-suited for investigating PTSD because DNA methylation (DNAm) encodes individual response to trauma and may play a key role in the immune dysregulation characteristic of PTSD pathophysiology. In the current study, we leveraged recent methodological advances to investigate sex-specific shifts in DNAm-based leukocyte composition that are associated with lifetime PTSD. We estimated leukocyte composition on a combined methylation array dataset (483 participants, ∼450k CpG sites) consisting of two civilian cohorts, the Detroit Neighborhood Health Study and Grady Trauma Project. Sex-stratified Mann-Whitney U test and two-way ANCOVA revealed that lifetime PTSD was associated with a small but significant elevation in monocyte proportions in males, but not in females (Holm-adjusted p-val < 0.05). No difference in monocyte proportions was observed between current and remitted PTSD cases in males, suggesting that this sex-specific peak shift reflects a long-standing trait of lifetime history of PTSD, rather than current state of PTSD. Associations with lifetime PTSD or PTSD status were not observed in any other leukocyte subtype and our finding in monocytes was confirmed using cell estimates based on a different deconvolution algorithm, suggesting that our sex-specific findings are robust across cell estimation approaches. Overall, our main finding of elevated monocyte proportions in males, but not in females with lifetime history of PTSD provides evidence for a sex-specific shift in peripheral blood leukocyte composition that is detectable in methylomic profiles and that reflects long-standing changes associated with PTSD diagnosis.
New non-bilaterian transcriptomes provide novel insights into the evolution of coralskeletomes
Authors: Conci Nicola , Wörheide Gert , Vargas Sergio
Published: April 2019

A general feature of animalskeletomesis the co-presence of taxonomically widespread and lineage-specific proteins that actively regulate the biomineralization process. Among cnidarians, the skeletomes of scleractinian corals have been shown to follow this trend, however in this group distribution and phylogenetic analyses of biomineralization-related genes have been often based on limited numbers of species, with other anthozoan calcifiers such as octocorals, being overlooked. Wede-novosequenced the transcriptomes of four soft-coral species characterized by different calcification strategies (aragonite skeletonvs. calcitic sclerites) and data-mined published non-bilaterian transcriptomic resources to construct a taxonomically comprehensive sequence database to map the distribution of scleractinian and octocoralskeletomecomponents. At the large scale, no protein showed a ‘Cnidaria+Placozoa’ or ‘Cnidaria+Ctenophora’ distribution, while some were found in cnidarians and Porifera. Within Scleractinia and Octocorallia, we expanded the distribution for several taxonomically restricted genes (TRGs) and propose an alternative evolutionary scenario, involving an early single biomineralization-recruitment event, for galaxinsensu stricto. Additionally, we show that the enrichment of acidic residues within skeletogenic proteins did not occur at the Corallimorpharia-Scleractinia transition, but appears to be associated with protein secretion in the organic matrix. Finally, the distribution of octocoral calcification-related proteins appears independent of skeleton mineralogy (i.e. aragonite/calcite) with no differences on the proportion of shared skeletogenic proteins between scleractinians and aragonitic or calcitic octocorals. This points toskeletomehomogeneity within but not between groups of calcifying cnidarians, although some proteins like galaxins and SCRiP-3a could represent instances of commonality.
A Ssd1 homolog impacts trehalose and chitin biosynthesis and contributes to virulence inAspergillus fumigatus
Authors: Thammahong Arsa , Dhingra Sourabh , Bultman Katherine M. , Kerkaert Joshua , Cramer Robert A.
Published: April 2019

Regulation of fungal cell wall biosynthesis is critical to maintain cell wall integrity in the face of dynamic fungal infection microenvironments. In this study, we observe that a yeastssd1homolog,ssdA,in the filamentous fungusAspergillus fumigatusis involved in trehalose and cell wall homeostasis. AnssdAnull mutant strain exhibited an increase in trehalose levels and a reduction in colony growth rate. Over-expression ofssdAin contrast perturbed trehalose biosynthesis and reduced conidia germination rates. ThessdAnull mutant strain was more resistant to cell wall perturbing agents while over-expression ofssdApromoted increased sensitivity. Over-expression ofssdAsignificantly increased chitin levels and both loss and over-expression ofssdAaltered sub-cellular localization of the class V chitin synthase CsmA. Strikingly, over-expression ofssdAabolished adherence to abiotic surfaces and severely attenuated the virulence ofA. fumigatusin a murine model of invasive pulmonary aspergillosis. In contrast, despite the severein vitrofitness defects observed upon loss ofssdA,neither surface adherence or murine survival was impacted. In conclusion,A. fumigatusSsdA plays a critical role in cell wall homeostasis that alters fungal-host interactions.

Importance

Life threatening infections caused by the filamentous fungusAspergillus fumigatusare increasing along with a rise in fungal strains resistant to contemporary antifungal therapies. The fungal cell wall and the associated carbohydrates required for its synthesis and maintenance are attractive drug targets given that many genes encoding proteins involved in cell wall biosynthesis and integrity are absent in humans. Importantly, genes and associated cell wall biosynthesis and homeostasis regulatory pathways remain to be fully defined inA. fumigatus.In this study, we identify SsdA, a model yeast Ssd1p homolog, as an important component of trehalose and fungal cell wall biosynthesis inA. fumigatusthat consequently impacts fungal virulence in animal models of infection.
An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min fromE. coliandM. smegmatiscells on a paper substrate
Authors: Naik Priyanka , Jaitpal Siddhant , Shetty Prasad , Paul Debjani
Published: April 2019

Developing sensors in the domains of food safety, soil analysis, water quality monitoring and healthcare often requires distinguishing between different species of bacteria. The most rapid, sensitive and specific method to identify bacteria is by analysing their DNA sequence, which comprises of disinfection and lysis of bacterial cells, amplification of the isolated DNA and detection of the amplified sequence. Seamless integration of these assays on a paper substrate remains a big challenge in paperfluidic nucleic acid analyis. Combining lysis and isothermal amplification in a single reaction step is difficult because the porosity of paper and the presence of cell debris following lysis reduces the efficiency of DNA amplification. On the other hand, extracting and purifying the DNA after lysis to improve the amplification efficiency involves addition of chemical reagents, one or more wash steps and manual intervention. This problem is even more complex for mycobacteria as its thick cell wall structure impedes lysis and the high GC-content of the genome requires careful optimization of enzymatic denaturation during isothermal amplification. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper without the need for any intermediate intervention. We demonstrate our integrated assay by amplifying DNA from 100 CFU/mL ofEscherichia coli(MG1655) andMycobacterium smegmatis(mc2155) cells in 30 min on a paper substrate. We also confirm thatE. coliandM. smegmatiscan be completely disinfected on paper by heating at 60 °C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.

Electronic Supplementary Information (ESI) is available.
A likelihood method for estimating present-day human contamination in ancient DNA samples using low-depth haploid chromosome data
Authors: Moreno-Mayar J. Víctor , Korneliussen Thorfinn Sand , Albrechtsen Anders , Dalal Jyoti , Renaud Gabriel , Nielsen Rasmus , Malaspinas Anna-Sapfo
Published: April 2019

1

Motivation

The presence of present-day human contaminating DNA fragments is one of the challenges defining ancient DNA (aDNA) research. This is especially relevant to the ancient human DNA field where it is difficult to distinguish endogenous molecules from human contaminants due to their genetic similarity. Recently, with the advent of high-throughput sequencing and new aDNA protocols, hundreds of ancient human genomes have become available. Contamination in those genomes has been measured with computational methods often developed specifically for these empirical studies. Consequently, some of these methods have not been implemented and tested while few are aimed at low-depth data, a common feature in aDNA datasets.

Results

We develop a new X-chromosome-based maximum likelihood method for estimating present-day human contamination in low-depth sequencing data. We implement our method for general use, assess its performance under conditions typical of ancient human DNA research, and compare it to previous nuclear data-based methods through extensive simulations. For low-depth data, we show that existing methods can produce unusable estimates or substantially underestimate contamination. In contrast, our method provides accurate estimates for a depth of coverage as low as 0.5× on the X-chromosome when contamination is below 25%. Moreover, our method still yields meaningful estimates in very challenging situations, i.e., when the contaminant and the target come from closely related populations or with increased error rates. With a running time below five minutes, our method is applicable to large scale aDNA genomic studies.

Availability and implementation

The method is implemented in C++ and R and is freely available in https://github.com/sapfo/contaminationX.

Contact

morenomayar@gmail.com, annasapfo.malaspinas@unil.ch.
Dual-matrix 3D culture system as a biomimetic model of epithelial tissues
Authors: Bogorodskaya Diana , McLane Joshua S. , Ligon Lee A.
Published: April 2019

Recent years have seen an unprecedented rise in the use of 3D culture systems, both in fundamental research and in more translational settings such as drug testing and disease modeling. However, 3D cultures often remain underused by cell biology labs, both due to technical difficulties in system setup and inherent drawbacks of many of the common systems. Here we describe an easy to use, inexpensive and rapidly assembled 3D culture system, suitable for generation of both normal polarized epithelial cysts and in-situ tumor spheroids. This system allows for exploration of many questions of normal and cancer cell biology, including morphogenesis, epithelial polarization, cell motility, intra- and intercellular communication, invasion, metastasis, and tumor-stoma interaction. The 3D cultures are made up of a stiffness tunable, dual-matrix model that can incorporate co-culture of multiple cell types. The model allows for increased physiological relevance by mimicking the organization, ligand composition and stiffness presentin-vivo. The setup allows for a wide spectrum of manipulation, including removing cells from the system for DNA/protein expression, transfection and high-resolution imaging of live or fixed cells.
Macrophage-mediated immunoediting drives ductal carcinoma evolution: Space is the game changer
Authors: Gatenbee Chandler , West Jeffrey , Baker Annie M. , Guljar Nafia , Jones Louise , Graham Trevor A. , Robertson-Tessi Mark , Anderson Alexander R. A.
Published: April 2019

Under normal conditions, the immune system is capable of rapidly detecting and eliminating potentially dangerous entities, including tumor cells. Due to intense selection pressure imposed by the immune response, tumor cells often evolve strategies to avoid elimination in a process known as immunoediting. It is less known how the evolutionary response to immune predation is altered by context. We explore the evolution of immune escape strategies in ductal cancers, a natural case in which to study evolution in different contexts: inside and outside of ducts. We highlight the role of macrophages as a source of “public goods,” releasing diffusible factors (reactive oxygen species and growth factors). Immunohistochemistry reveals differences between macrophage densities of invasive ductal carcinomas and non-invasive ductal carcinomasin situ. For the first time, immunohistochemistry (IHC) imaging data comparing DCIS to IDC were used to initialize mechanistic agent-based models of evolutionary dynamics. By using IHC to map the initial conditions of a growing tumor, we show that spatial competition and structure influence transient dynamics during invasion. These dynamics are context-dependent, a conclusion that may be missed from interpreting imaging or non-spatial modeling alone. Before invasion, the presence of macrophages correlate with shorter ductal breach times. After invasion, tumors may employ a “pioneer-engineer” strategy where pioneering immunoresistant cells on the tumor’s edge stimulate the release of M1-macrophage-derived reactive oxygen species, degrading surrounding stroma. Behind the invasive edge, the engineering immunosuppressive cells promote the release of M2-macrophage-derived growth factors, providing a long-term immune escape strategy. Together, mathematical modeling and image analysis highlight the crucial role tumor-associated macrophages play in immune escape and invasion, both inside and outside of ducts.
Latitudinal variation in circadian rhythmicity in Nasonia vitripennis
Authors: Paolucci Silvia , Benetta Elena Dalla , Salis Lucia , Doležel David , van de Zande Louis , Beukeboom Leo W.
Published: April 2019

Many physiological processes of living organisms show circadian rhythms, governed by an endogenous clock. This clock has a genetic basis and is entrained by external cues such as light and temperature. Other physiological processes exhibit seasonal rhythms, that are also responsive to light and temperature. We previously reported a natural latitudinal cline of photoperiodic diapause induction in the parasitic wasp Nasonia vitripennis in Europe and a correlated haplotype frequency for the circadian clock gene period (per). To evaluate if this correlation is reflected in circadian behaviour, we investigated circadian locomotor activity of seven populations from the cline. We found that the proportion of rhythmic males is higher than females in constant darkness, and that mating decreased rhythmicity of both sexes. Only for virgin females, the free running period (τ) increased weakly with latitude. Wasps from the most southern locality had an overall shorter free running rhythm and earlier onset, peak and offset of activity during the 24 h period, than wasps from the northernmost locality. We evaluate this variation in rhythmicity as a function of period haplotype frequencies in the populations and discuss its functional significance in the context of local adaptation.
Modern clinical Mycobacterium tuberculosis strains leverage type I IFN pathway for a pro-inflammatory response in the host
Authors: Shankaran Deepthi , Arumugam Prabhakar , Bothra Ankur , Gandotra Sheetal , Rao Vivek
Published: April 2019

Host phagocytes respond to infections by innate defense mechanisms through metabolic shuffling in order to restrict the invading pathogen. However, this very plasticity of the host provides an ideal platform for pathogen mediated manipulation. By employing the macrophage model of Mtb infection, we identify an important strategy employed by modern clinical lineages in regulating the host immune–metabolism axis. The potent ability of these strains to specifically elicit a strong and early macrophage type I IFN response (in contrast to the protracted response to ancient Mtb), was dependent on an increased ability to localize in acidified phagosomes; this higher transit via acidified compartments is important for stimulation of the DNA dependent signaling in infected macrophages. The augmented IFN signaling provided a positive regulatory loop for enhanced expression of the cellular oxysterol-CH25H which in turn facilitated higher levels of IL6 in macrophages infected with the modern Mtb strains. Requirement of type I IFN signaling in mycobacterial intracellular growth highlights another unique ability of Mtb to manipulate host cell physiology and proinflammatory responses.

Significance Statement

Co-evolution with humans has enabled the development of novel adaptive mechanisms for survival in host specific environments in the human TB pathogen-Mtb. We present one such mechanism of modern Mtb strains harnessing the type I IFN immune axis to regulate the host pro-inflammatory response. Our results highlight the use of host intracellular endosomal transit as a mechanism by these strains to ensure a strong type I IFN response in macrophages. We also demonstrate the ability of Mtb to regulate macrophage cholesterol metabolism in order to fine tune the host innate responses. These findings lay the foundation of the future development of a host axis directed intervention strategy against this pathogen.
Axonal Transport Of An Insulin-Like Peptide Mrna Promotes Stress Recovery InC. Elegans
Authors: Chandra Rashmi , Li Lisa , Husain Zahabiya , Mishra Shashwat , Alcedo Joy
Published: April 2019

Aberrations in insulin or insulin-like peptide (ILP) signaling in the brain causes many neurological diseases. Here we report that mRNAs of specific ILPs are surprisingly mobilized to the axons ofC. elegansduring stress. Transport of the ILPins-6mRNA to axons facilitates recovery from stress, whereas loss of axonal mRNA delays recovery. In addition, the axonal traffic ofins-6mRNA is regulated by at least two opposing signals: one that depends on the insulin receptor DAF-2 and a kinesin-2 motor; and a second signal that is independent of DAF-2, but involves a kinesin-3 motor. While Golgi bodies that package nascent peptides, like ILPs, have not been previously found inC. elegansaxons, we show that axons of stressedC. eleganshave increased Golgi ready to package peptides for secretion. Thus, our findings present a mechanism that facilitates an animal’s rapid recovery from stress through axonal ILP mRNA mobilization.
Functional Mature Human Microglia Developed in Human iPSC Microglial Chimeric Mouse Brain
Authors: Xu Ranjie , Boreland Andrew J. , Li Xiaoxi , Posyton Anthony , Kwan Kelvin , Hart Ronald P. , Jiang Peng
Published: April 2019

Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under homeostatic states. We developed a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage precursors into neonatal mouse brains. The engrafted human microglia widely disperse in the brain and replace mouse microglia in corpus callosum at 6 months post-transplantation. Single-cell RNA-sequencing of the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a novel tool to study the role of human microglia in brain development and degeneration.
A post-translational modification signature defines changes in soluble tau correlating with oligomerization in early stage Alzheimer’s disease brain
Authors: Ercan-Herbst Ebru , Schöndorf David C. , Behrendt Annika , Klaus Bernd , Ramos Borja Gomez , Weber Christian , Ehrnhoefer Dagmar E.
Published: April 2019

Tau is a microtubule-binding protein that can receive various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Hyperphosphorylation of tau is linked to its aggregation and the formation of neurofibrillary tangles (NFTs), which are a hallmark of Alzheimer’s disease (AD). While more than 70 phosphorylation sites have been detected previously on NFT tau, studies of oligomeric and detergent-soluble tau in human brains during the early stages of AD are lacking. Here we apply a comprehensive electrochemiluminescence ELISA assay to analyze twenty-five different PTM sites as well as tau oligomerization in control and sporadic AD brain. The samples were classified as Braak stages 0-I, II or III-IV, respectively, corresponding to the progression of microscopically detectable tau pathology throughout different brain regions. We find that soluble tau oligomers are strongly increased at Braak stages III-IV in all brain regions under investigation, including the temporal cortex, which does not contain NFTs at this stage of pathology. We additionally identified five phosphorylation sites that are specifically and consistently increased across the entorhinal cortex, hippocampus and temporal cortex in the same donors. Three of these sites correlate with tau oligomerization in all three brain regions, but do not overlap with the epitopes of phospho-sensitive antibodies commonly used for the immunohistochemical detection of NFTs. Our results thus suggest that soluble oligomers are characterized by a small set of specific phosphorylation events that differ from those dominating in mature NFTs and shed light on early PTM changes of tau during AD pathogenesis in human brains.
Drosophilafemale-specific brain neuron elicits persistent position- and direction-selective male-like social behaviors
Authors: Wu Yang , Bidaye Salil S. , Mahringer David
Published: April 2019

Latent neural circuitry in the female brain encoding male-like mating behaviors has been revealed in both mice and flies. InDrosophila, a key component of this circuitry consists of thedoublesex-expressing pC1 neurons, which were deemed to exist in both sexes and function based on the amount of cells being activated. Here, we identify pC1-alpha, a female-specific subtype of pC1, as responsible for inducing persistent male-like social behaviors in females. We demonstrate that activation of a single pC1-alpha neuron is sufficient for such induction in a position- and direction-selective manner, and activity of pC1-alpha neurons as a whole is indispensable for maintaining normal sexual receptivity. These dual functions of pC1-alpha may require different neurotransmission, with acetylcholine specifically required for the former but not the latter. Our findings suggest that pC1-alpha may be the female counterpart of male P1 due to their shared similarities in morphology, lineage, and social promoting function.
Mycobacterium tuberculosis cording in the cytosol of live lymphatic endothelial cells
Authors: Lerner Thomas R. , Queval Christophe J. , Lai Rachel P. , Russell Matthew , Fearns Antony , Greenwood Daniel J. , Collinson Lucy , Wilkinson Robert J. , Gutierrez Maximiliano G.
Published: April 2019

The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis remains obscure. We show that several M. tuberculosis clinical isolates form intracellular cords in primary human lymphatic endothelial cells (hLEC) in vitro and also in the lymph nodes of patients with tuberculosis. We identified via RNA-seq a transcriptional programme in hLEC that activates cellular pro-survival and cytosolic surveillance of intracellular pathogens pathways. Consistent with this, cytosolic access of hLEC is required for intracellular M. tuberculosis cording; and cord formation is dependent on the M. tuberculosis ESX-1 type VII secretion system and the mycobacterial lipid PDIM. Finally, we show that M. tuberculosis cording is a novel size-dependent mechanism used by the pathogen to evade xenophagy in the cytosol of endothelial cells. These results provide a mechanism that explains the long-standing association between M. tuberculosis cording and virulence.