bluuebunny/crossref_metadata_2025 huggingface dataset:
Stylistic palimpsests: Computational stylistic perspectives on precursory authorship in Aphra Behn’s drama
Authors: Evans Mel , Hogarth Alan
Published: December 2019

Computational stylistics has developed various methods for investigating and attributing authorship of collaborative literary texts. This article investigates ‘precursory authorship’ (Love, 2002): that is, the authorial traces of a source text that inform—to a greater or lesser degree—a subsequent literary output, in order to establish its relevance for our approach to and understanding of the linguistic properties of literary style. Precursory authorship and derivative adaptations are common features of early modern English drama, and the study focusses on two case studies relating to the plays of Restoration playwright, Aphra Behn (c. 1640–89). Using a combination of quantitative methods (Rolling Delta (RD), principal components analysis (PCA), Delta, and Hierarchical Cluster Analysis), the investigation highlights the presence of precursory authorial style in Behn’s The Rover and an anonymous work associated with Behn, The Counterfeit Bridegroom. The results suggest that precursory authorial style is identifiable in both cases, not only through a similarity with the source text but, to a lesser degree, other texts by the precursory author as well. The anonymous play yields complex and non-confirmatory evidence for Behn’s authorship. Methodologically, RD is most sensitive to precursory collaboration. Collectively, the findings highlight the importance of stylistic factors when describing and interpreting literary linguistic quantitative data: precursory authorial style is another facet that intersects with properties such as time period and genre. The article urges a more critical and theoretically informed view of authorially aligned linguistic style.
Cardiovascular risk of nonsteroidal anti-inflammatory drugs in dialysis patients: a nationwide population-based study
Authors: Jo Hyung Ah , Kim Dong Ki , Park Seokwoo , Kim Yaerim , Han Seung Seok , Yang Bo Ram , Choi So-Hyun , Kim Mi-Sook , Lee Joongyub , Lee Hajeong , Lee Jung Pyo , Lim Chun Soo , Kim Yon Su , Joo Kwon Wook
Published: December 2019

Background

Given the cardiovascular risk of nonsteroidal anti-inflammatory drugs (NSAIDs), it is essential to identify the relationship between NSAIDs and cardiovascular outcomes in dialysis patients who have elevated cardiovascular risk.

Methods

A case-crossover study was conducted to assess the association of NSAIDs with major adverse cardiac and cerebrovascular events (MACCEs) and mortality using the Korean Health Insurance dataset. The case period was defined as 1–30 days prior to the event date and the control periods were defined as 61–90 days and 91–120 days prior to the event date.

Results

There were 3433 and 8524 incident dialysis patients who experienced MACCEs and mortality, respectively, after exposure to NSAIDs within 120 days before each event. NSAIDs significantly increased the risk of MACCEs {adjusted odds ratio [aOR] 1.37 [95% confidence interval (CI) 1.26–1.50]} and mortality [aOR 1.29 (95% CI 1.22–1.36)]. Nonselective NSAIDs, but not selective cyclooxygenase-2 inhibitors, significantly increased the risk of MACCEs and mortality. However, the MACCE and mortality risk did not increase in a dose-dependent manner in the analysis according to the cumulative defined daily dosage of NSAIDs. The incidence of MACCEs in the case period tended to be more common in patients who had recent exposure to NSAIDs than in patients who did not have recent exposure to NSAIDs.

Conclusions

Clinicians should be particularly cautious when prescribing NSAIDs to dialysis patients considering the associations of NSAIDs with cardiovascular outcomes and mortality, which might occur independent of the dose and duration of exposure.
Transversely trapping surfaces: Dynamical version
Authors: Yoshino Hirotaka , Izumi Keisuke , Shiromizu Tetsuya , Tomikawa Yoshimune
Published: December 2019

We propose new concepts, a dynamically transversely trapping surface (DTTS) and a marginally DTTS, as indicators for a strong gravity region. A DTTS is defined as a two-dimensional closed surface on a spacelike hypersurface such that photons emitted from arbitrary points on it in transverse directions are acceleratedly contracted in time, and a marginally DTTS is reduced to the photon sphere in spherically symmetric cases. (Marginally) DTTSs have a close analogy with (marginally) trapped surfaces in many aspects. After preparing the method of solving for a marginally DTTS in the time-symmetric initial data and the momentarily stationary axisymmetric initial data, some examples of marginally DTTSs are numerically constructed for systems of two black holes in the Brill–Lindquist initial data and in the Majumdar–Papapetrou spacetimes. Furthermore, the area of a DTTS is proved to satisfy the Penrose-like inequality $A_0\le 4\pi (3GM)^2$, under some assumptions. Differences and connections between a DTTS and the other two concepts proposed by us previously, a loosely trapped surface [Prog. Theor. Exp. Phys. 2017, 033E01 (2017)] and a static/stationary transversely trapping surface [Prog. Theor. Exp. Phys. 2017, 063E01 (2017)], are also discussed. A (marginally) DTTS provides us with a theoretical tool to significantly advance our understanding of strong gravity fields. Also, since DTTSs are located outside the event horizon, they could possibly be related with future observations of strong gravity regions in dynamical evolutions.
Myofascia-dominant involvement on whole-body MRI as a risk factor for rapidly progressive interstitial lung disease in dermatomyositis
Authors: Karino Kohei , Kono Michihiro , Kono Michihito , Sakamoto Keita , Fujieda Yuichiro , Kato Masaru , Amengual Olga , Oku Kenji , Yasuda Shinsuke , Atsumi Tatsuya
Published: December 2019

Objective

Rapidly progressive interstitial lung disease (RPILD) is a major cause of death in patients with DM. Although clinically amyopathic DM (CADM) represents risk for RPILD, the incidence rate of RPILD in patients with CADM varies widely. Whole-body (WB) MRI can reveal involvement of systemic muscle and myofascia. The objective of this study was to explore the risk factors for RPILD in patients with DM using WB-MRI.

Methods

This retrospective study comprised 41 patients with DM who underwent WB-MRI before the initiation of treatment in our hospital. Muscular and myofascial signals were scored on 42 muscular groups. The myofascia/muscle ratio was calculated and used to define the relevance of myofascia-dominant involvement. RPILD was defined as worsening of dyspnoea, hypoxaemia and radiographic ILD/fibrosis within 3 months from the onset of respiratory symptoms.

Results

Among the 41 patients, 17 had CADM and 30 had ILD, including 10 patients with RPILD. All patients including those with CADM showed abnormal signal intensity in both muscle and myofascia (median score: 15 and 23, respectively). Muscle signal scores positively correlated with the serum creatine kinase level (r = 0.714; P< 0.001). Patients with RPILD showed a significantly higher myofascia/muscle ratio than those without RPILD (1.929 vs 1.200; P= 0.027). Logistic regression analysis identified higher myofascia/muscle ratio as independent risk factors for developing RPILD.

Conclusion

Myofascia-dominant involvement was defined and appreciated in patients with DM using WB-MRI. This may be one of the risk factors for RPILD.
Network outcome analysis identifies difficulty initiating sleep as a primary target for prevention of depression: a 6-year prospective study
Authors: Blanken Tessa F , Borsboom Denny , Penninx Brenda Wjh , Van Someren Eus Jw
Published: December 2019

Study Objectives

Major depressive disorder (MDD) is the leading cause of disability worldwide. Its high recurrence rate calls for prevention of first-onset MDD. Although meta-analysis suggested insomnia as the strongest modifiable risk factor, previous studies insufficiently addressed that insomnia might also occur as a residual symptom of unassessed prior depression, or as a comorbid complaint secondary to other depression risks.

Methods

In total, 768 participants from the Netherlands Study of Depression and Anxiety who were free from current and lifetime MDD were followed-up for four repeated assessments, spanning 6 years in total. We performed separate Cox proportional hazard analyses to evaluate whether baseline insomnia severity, short-sleep duration, and individual insomnia complaints prospectively predicted first-onset MDD during follow-up. The novel method of network outcome analysis (NOA) allowed us to sort out whether there is any direct predictive value of individual insomnia complaints among several other complaints that are associated with insomnia.

Results

Over 6-year follow-up, 141 (18.4%) were diagnosed with first-onset MDD. Insomnia severity but not sleep duration predicted first-onset MDD (HR = 1.11, 95% CI: 1.07–1.15), and this was driven solely by the insomnia complaint difficulty initiating sleep (DIS) (HR = 1.10, 95% CI: 1.04–1.16). NOA likewise identified DIS only to directly predict first-onset MDD, independent of four other associated depression complaints.

Conclusions

We showed prospectively that DIS is a risk factor for first-onset MDD. Among the different other insomnia symptoms, the specific treatment of DIS might be the most sensible target to combat the global burden of depression through prevention.
Sex differences within sleep in gonadally intact rats
Authors: Swift Kevin M , Keus Karina , Echeverria Christy Gonzalez , Cabrera Yesenia , Jimenez Janelly , Holloway Jasmine , Clawson Brittany C , Poe Gina R
Published: December 2019

Sleep impacts diverse physiological and neural processes and is itself affected by the menstrual cycle; however, few studies have examined the effects of the estrous cycle on sleep in rodents. Studies of disease mechanisms in females therefore lack critical information regarding estrous cycle influences on relevant sleep characteristics. We recorded electroencephalographic (EEG) activity from multiple brain regions to assess sleep states as well as sleep traits such as spectral power and interregional spectral coherence in freely cycling females across the estrous cycle and compared with males. Our findings show that the high hormone phase of proestrus decreases the amount of nonrapid eye movement (NREM) sleep and rapid eye movement (REM) sleep and increases the amount of time spent awake compared with other estrous phases and to males. This spontaneous sleep deprivation of proestrus was followed by a sleep rebound in estrus which increased NREM and REM sleep. In proestrus, spectral power increased in the delta (0.5–4 Hz) and the gamma (30–60 Hz) ranges during NREM sleep, and increased in the theta range (5–9 Hz) during REM sleep during both proestrus and estrus. Slow-wave activity (SWA) and cortical sleep spindle density also increased in NREM sleep during proestrus. Finally, interregional NREM and REM spectral coherence increased during proestrus. This work demonstrates that the estrous cycle affects more facets of sleep than previously thought and reveals both sex differences in features of the sleep–wake cycle related to estrous phase that likely impact the myriad physiological processes influenced by sleep.
Overcoming cultural barriers to implementing oral HIV self-testing with high fidelity among Tanzanian youth
Authors: Catania Joseph A , Huun Cassidy , Dolcini M Margaret , Urban Angelmary Joel , Fleury Nick , Ndyetabula Clinton , Singh Ryan , Young Amy W , Conserve Donaldson F , Lace James , Msigwa Joyce Samweli
Published: December 2019

Tanzanian youth have high levels of HIV risk and poor access to HIV-testing. Oral self-implemented testing (Oral-SIT) provides an alternative that reduces barriers to HIV-testing. We examined adaptations to Oral-SIT training components in a randomized experiment to evaluate a “train-the-trainer” strategy for improving comprehension of graphic training materials. Participants (N = 257, age = 14−19 years) were randomly assigned to one of two self-training conditions: graphic instruction book (GIB) or Video-GIB. Outcomes included behavioral performance fidelity, self-reported comprehension, and intentions to seek treatment. Video-GIB participants, relative to GIB-only participants, had higher performance fidelity scores, made fewer performance errors, had better instruction comprehension, and were more likely to intend to seek treatment. Oral-SIT timing errors were significantly more common among GIB-only participants.

Graphic training materials in conjunction with a “train-the-trainer” video has significant potential for increasing Oral-SIT’s reach by overcoming technological and literacy barriers.
Acute Skin Failure in the Critical Care Patient
Authors: Delmore Barbara , Cox Jill , Smith Daniel , Chu Andy S. , Rolnitzky Linda
Published: December 2019

OBJECTIVE

The purpose of this research was to build on previous work regarding predictive factors of acute skin failure (ASF) in the critically ill population.

METHODS

Researchers conducted a retrospective case-control study with a main and validation analysis. Data were extracted from the New York Statewide Planning and Research Cooperative System. For the main analysis, there were 415 cases with a hospital-acquired pressure injury (HAPI) and 194,872 controls without. Researchers then randomly selected 100 cases with a HAPIs and 300 controls without for the validation analysis. A step-up logistic regression model was used. Researchers generated receiver operating characteristic curves for both the main and validation analyses, assessing the overall utility of the regression model.

RESULTS

Eleven variables were significantly and independently related to ASF: renal failure (odds ratio [OR], 1.4, P = .003), respiratory failure (OR, 2.2; P = < .001), arterial disease (OR, 2.4; P = .001), impaired nutrition (OR, 2.3; P = < .001), sepsis (OR, 2.2; P = < .001), septic shock (OR, 2.3; P = < .001), mechanical ventilation (OR, 2.5; P = < .001), vascular surgery (OR, 2.2; P = .02), orthopedic surgery (OR, 3.4; P = < .001), peripheral necrosis (OR, 2.5; P = .003), and general surgery (OR, 3.8; P = < .001). The areas under the curve for the main and validation analyses were 0.864 and 0.861, respectively.

CONCLUSIONS

The final model supports previous work and is consistent with the current definition of ASF in the setting of critical illness.
The Positivism Paradigm of Research
Authors: Park Yoon Soo , Konge Lars , Artino Anthony R.
Published: December 2019

Research paradigms guide scientific discoveries through their assumptions and principles. Understanding paradigm-specific assumptions helps illuminate the quality of findings that support scientific studies and identify gaps in generating sound evidence. This article focuses on the research paradigm of positivism, examining its definition, history, and assumptions (ontology, epistemology, axiology, methodology, and rigor). Positivism is aligned with the hypothetico-deductive model of science that builds on verifying a priori hypotheses and experimentation by operationalizing variables and measures; results from hypothesis testing are used to inform and advance science. Studies aligned with positivism generally focus on identifying explanatory associations or causal relationships through quantitative approaches, where empirically based findings from large sample sizes are favored—in this regard, generalizable inferences, replication of findings, and controlled experimentation have been principles guiding positivist science. Criteria for evaluating the quality of positivist research are discussed. An example from health professions education is provided to guide positivist thinking in study design and implementation.
Angiotensin–neprilysin inhibition confers renoprotection in rats with diabetes and hypertension by limiting podocyte injury
Authors: Uijl Estrellita , ‘t Hart Daan C. , Roksnoer Lodi C.W. , Groningen Marian C. Clahsen-van , van Veghel Richard , Garrelds Ingrid M. , de Vries René , van der Vlag Johan , Zietse Robert , Nijenhuis Tom , Joles Jaap A. , Hoorn Ewout J. , Danser A.H. Jan
Published: December 2019

Objectives:

Combined angiotensin receptor--neprilysin inhibition (ARNI) reduces glomerulosclerosis better than single angiotensin receptor blockade (ARB) in diabetic, hypertensive rats. The renoprotective mechanism remains unknown, but may depend on superior blood pressure control, improved renal hemodynamics, suppressed renal inflammation or prevention of podocyte loss.

Methods:

To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic for 12 weeks and treated with vehicle (n = 10), valsartan (ARB; n = 7) or sacubitril/valsartan (ARNI; n = 8) for the final 3 weeks. Arterial pressure was measured via radiotelemetry.

Results:

Sacubitril/valsartan lowered mean arterial pressure by −50 ± 4 mmHg and valsartan by −43 ± 4 mmHg (P = 0.3). Both treatments lowered albuminuria, but only sacubitril/valsartan maintained high urinary atrial natriuretic peptide, improved glycemic control and protected podocyte integrity, reflected by increased nephrin expression and suppression of transient receptor potential canonical 6 and regulator of calcineurin 1. This resulted in markedly reduced glomerulosclerosis (P < 0.05 vs. control and valsartan). Despite higher effective renal plasma flow and glomerular filtration rates, sacubitril/valsartan did neither improve filtration fraction nor renal immune cell infiltration.

Conclusion:

Sacubitril/valsartan offers drug-class-specific renoprotection in a preclinical model of diabetes and hypertension. Renoprotection is unrelated to antihypertensive efficacy, renal hemodynamics or inflammation, but may be related to protective effects of natriuretic peptides on podocyte integrity.
Drug-Induced Extrapyramidal Symptoms at the Pediatric Emergency Department
Authors: Chang Ming-Yu , Lin Kuang-Lin , Wang Huei-Shong , Wu Chang-Teng
Published: December 2019

Objectives

Extrapyramidal symptoms (EPS) induced by pharmacologic agents can cause patient discomfort and lead to emergency department visits. Analyzing these cases at a pediatric emergency department may help to elucidate the characteristic features of extrapyramidal syndrome in children.

Methods

This retrospective study was conducted at Chang Gung Memorial Hospital in Taiwan. Pediatric patients with drug-induced extrapyramidal syndrome seeking treatment at our emergency department from January 2001 to December 2010 were enrolled. The patients' clinical features, drug history, demographic data, and treatment data were collected and analyzed.

Results

One hundred nineteen patients (61 females, 58 males) were enrolled. Ninety-six patients could provide their drug history; all of whom took dopamine antagonists and 90% of whom took dopamine antagonists as antiemetic agents, with only 9 patients taking them for antipsychotic purposes. Metoclopramide syrup overdose was the main cause of extrapyramidal syndrome in patients under 2 years old. The average emergency room stay of the patients who could provide their drug history was shorter than that of those who could not.

Conclusions

It is not uncommon for patients with drug-induced EPS to present to a pediatric emergency room owing to the use of dopamine antagonists as antiemetic agents. Clinical symptoms with a clear drug history are helpful for the diagnosis and management. Emphasizing the correct usage of liquid medications will reduce the risk of EPS.
Incidence and Imminence of Pregnancy in At-Risk Teens After Emergency Department Visits
Authors: Smith Alla Lescure , Chen Lei
Published: December 2019

Background

Interventions aimed at reducing teen pregnancy rates in the United States have focused on clinics and schools. Teens disproportionately seek care in emergency departments (EDs), making these an important and understudied arena for interventions to prevent subsequent unwanted pregnancies. Establishing the risk of subsequent pregnancy (SP) in this population is a necessary prelude to effective interventions. Therefore, we set out to measure the incidence and imminence of pregnancy in sexually active teens after an ED visit.

Methods

A medical record review was conducted in an urban medical center with a general ED and a pediatric ED. Subjects were included if they were female individuals aged 13 to 19 years, were tested for gonorrhea and chlamydia in the EDs from 2004 to 2006, and were patients in the primary care clinics at the affiliated institution. Subsequent pregnancies were determined from the primary clinic charts. The duration of follow-up was 4 years.

Results

Three hundred ninety-eight subjects were included in the study. The mean age at ED visit was 17.3 years. A majority (70.1%) had a documented SP. For patients with an SP, the mean interval from ED visit to conception was 15.8 months. Patients who had an SP were significantly more likely to be an ethnic minority, to have tested positive for gonorrhea, and to have visited the adult ED.

Conclusions

In this population of sexually active teens, 70.1% became pregnant within 4 years of being tested for gonorrhea and chlamydia in the ED. The encounter in the ED represents a potential opportunity for pregnancy-prevention interventions.
Improving Resident Well-Being During Shiftwork
Authors: Levin Helen , Lim Rodrick , Lynch Timothy , Sangha Gurinder
Published: December 2019

Purpose

The objective of this study is to quantitatively evaluate the well-being of residents doing casino shifts compared with those doing standard overnight shifts while working in an academic pediatric emergency department.

Methods

A randomized prospective survey study was performed over a period of 1 year on all residents who were scheduled to complete a 28-day block. Each block (28-day period) within the year was designated as either a “standard” or “casino” block. The standard overnight shifts were scheduled from midnight to 0800 hours, and casino shifts occurred from either 2000 to 0400 hours (casino A) and 0400 to 1200 hours (casino B). Participating residents were asked to complete both a preblock and postblock survey. The primary outcome was defined as differences in resident well-being as assessed by the brief resident wellness profile (BRWP). A mood faces graphical rating item to assess overall mood was used as a secondary outcome measures as well as a 10-item survey based on World Health Organization domains for quality of life and adapted to reflect completion of shiftwork.

Results

A total of 124 (90%) of 138 residents completed the study and were included in the analysis. No significant difference in resident well-being measured by BRWP between those in the standard and casino shift groups (17 ± 2.5 for preblock standard and 16.9 ± 2.8 for casino, P = 0.904; 17.1 ± 2.7 for postblock standard and 17.2 ± 3.1 for casino, P = 0.817), or in the relative change of the BRWP preblock and postblock between the 2 groups (standard, 0.35 ± 2.7; casino, 0.29 ± 3.0; P = 0.926). No significant difference in the mood faces rating scale scores or the 10-item postblock survey was found.

Conclusions

In the first study examining the effects of casino shifts on trainees, we found no effect of standard overnight versus casino shifts on their well-being. This counters the benefits previously seen in emergency department consultant staff and highlights the need for more studies specifically in trainees.
Hippocampal Sulcus Remnant: Common Finding in Nonelderly Adults on Ultra-High-Resolution 7T Magnetic Resonance Imaging
Authors: Yamashita Koji , Zong Xiaopeng , Hung Sheng-Che , Lin Weili , Castillo Mauricio
Published: December 2019

Objective

The objective of this study was to investigate the frequency of hippocampal sulcus remnants (HSRs) in nonelderly adults using ultra-high-resolution 7T magnetic resonance (MR) images and their imaging features.

Methods

A total of 33 healthy adults underwent 7T MR, and multiplanar images of 66 temporal lobes were reviewed independently by 2 neuroradiologists. The detectability of the HSR was calculated. In addition, the interobserver agreement on the rating scale was evaluated using the κ statistic.

Results

Both observers identified HSRs with 7T MR images in all subjects. Excellent interobserver agreement was shown (κ = 1.0). The shape of HSRs was variable (spot-like, curvilinear, ovoid, or beaded appearance). Volumes of the HSRs were not correlated with age.

Conclusions

Hippocampal sulcus remnants are commonly seen in healthy nonelderly adults using 7T MR imaging. Accurate diagnosis of HSR based on the microanatomy of hippocampus makes it easier to differentiate them from lesions, and it may help prevent unnecessary treatment.
Organellar protein multi-functionality and phenotypic plasticity in plants
Authors: Mackenzie Sally A. , Kundariya Hardik
Published: December 2019

With the increasing impact of climate instability on agricultural and ecological systems has come a heightened sense of urgency to understand plant adaptation mechanisms in more detail. Plant species have a remarkable ability to disperse their progeny to a wide range of environments, demonstrating extraordinary resiliency mechanisms that incorporate epigenetics and transgenerational stability. Surprisingly, some of the underlying versatility of plants to adapt to abiotic and biotic stress emerges from the neofunctionalization of organelles and organellar proteins. We describe evidence of possible plastid specialization and multi-functional organellar protein features that serve to enhance plant phenotypic plasticity. These features appear to rely on, for example, spatio-temporal regulation of plastid composition, and unusual interorganellar protein targeting and retrograde signalling features that facilitate multi-functionalization. Although we report in detail on three such specializations, involving MSH1, WHIRLY1 and CUE1 proteins inArabidopsis, there is ample reason to believe that these represent only a fraction of what is yet to be discovered as we begin to elaborate cross-species diversity. Recent observations suggest that plant proteins previously defined in one context may soon be rediscovered in new roles and that much more detailed investigation of proteins that show subcellular multi-targeting may be warranted.

This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.
Impact of mitonuclear interactions on life-history responses to diet
Authors: Camus M. Florencia , O'Leary Michael , Reuter Max , Lane Nick
Published: December 2019

Mitochondria are central to both energy metabolism and biosynthesis. Mitochondrial function could therefore influence resource allocation. Critically, mitochondrial function depends on interactions between proteins encoded by the mitochondrial and nuclear genomes. Severe incompatibilities between these genomes can have pervasive effects on both fitness and longevity. How milder deficits in mitochondrial function affect life-history trade-offs is less well understood. Here, we analyse how mitonuclear interactions affect the trade-off between fecundity and longevity in
Drosophila melanogaster
. We consider a panel of 10 different mitochondrial DNA haplotypes against two contrasting nuclear backgrounds (
w
1118
(WE) and Zim53 (ZIM)) in response to high-protein versus standard diet. We report strikingly different responses between the two nuclear backgrounds. WE females have higher fecundity and decreased longevity on high protein. ZIM females have much greater fecundity and shorter lifespan than WE flies on standard diet. High protein doubled their fecundity with no effect on longevity. Mitochondrial haplotype reflected nuclear life-history trade-offs, with a negative correlation between longevity and fecundity in WE flies and no correlation in ZIM flies. Mitonuclear interactions had substantial effects but did not reflect genetic distance between mitochondrial haplotypes. We conclude that mitonuclear interactions can have significant impact on life-history trade-offs, but their effects are not predictable by relatedness.

This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.
Engineering synthetic mini-chromosomes to study chromosome segregation in budding yeast
Authors: Jonsson Katarina , Zulkower Valentin , Robertson Daniel , Cai Yizhi , Marston Adele
Published: December 2019

Chromosomes need to be replicated and partitioned into daughter cells during cellular division. Failure to do so leads to aneuploidy, which has been linked to medical conditions such as cancer. Cohesin, a ring-shaped protein complex, holds the replicated sister chromatids together to ensure their accurate segregation. In Saccharomyces cerevisiae a large pool of cohesin is loaded at centromeres. Introducing the centromeric sequence to circular and linear DNA has been exploited to allow propagation of extra-chromosomal DNA. However, these extra-chromosomal DNA do not segregate as well as endogenous chromosomes and the reasons for this are unknown. Interestingly, small circular chromosomes are more stable than their linear counterparts. Furthermore, the stability of linear chromosomes increases with chromosome length. The DNA sequence itself appears to be important as linear chromosomes containing endogenous DNA are more stable than chromosomes derived from artificial DNA of similar lengths. During our project, we sought to identify cis-acting DNA elements, other than the centromere, that are important for chromosome segregation.

We have developed a synthetic biology approach to engineer a library of synthetic circular and linear chromosomes of various lengths containing unnatural DNA (designed to lack features of known elements such as transcription units). These chromosomes act as a “blank canvas” where candidate DNA elements are introduced to test their functionality. Our design, build and test cycle has allowed us to determine how chromosome length, circularization and DNA sequence influence chromosome segregation and the minimal requirements for building the optimal synthetic chromosome. Our latest findings will be presented.
The activity of S. pombe LTR retrotransposons is activated in response to rapamycin
Authors: Chan Tsun Ho , Whitehall Simon
Published: December 2019

Long terminal repeat (LTR) retrotransposons are mobile genetic elements that are present in the genomes of most eukaryotes. They are closely related to retroviruses and mobilize through an RNA intermediate. The uncontrolled mobilization of retrotransposons is potentially harmful to the integrity of the genome and so the activity of these elements is subjected to strict host cell controls. We are using the fission yeast, Schizosaccharomyces pombe to study the signalling pathways that regulate the activity ofLTR retrotransposons. We have found that the expression and mobilization of the Tf2 LTR retrotransposons is activated in response to exposure to the immunosuppressant drug rapamycin. Rapamycin binds to the conserved FKBP12 protein (called Fkh1 in S. pombe) and the resulting FKBP12-rapamycin complex inhibits the kinase activity of the conserved the TORC1 complex. This suggests that Tf2 activity is under the control of the TORC1 signalling network which is a master regulator of cellular responses to nutrient and energy availability. However, the inhibition of TORC1 activity using a tor2 temperature sensitive allele or a direct chemical inhibitor (Torin) did not activate either the expression or mobilization of Tf2 elements. Therefore, rapamycin may be controlling Tf2 activity via a TORC1-independent pathway. We are currently defining this pathway and find that it is dependent upon the FKBP12 protein, Fkh1 and the Forkhead transcription factor, Fhl1.
Inhibition of Drug-Resistant Candida glabrata by Killer Toxins Produced by Saccharomyces cerevisiae
Authors: Fredericks Lance , Roslund Cooper , Crabtree Angela , Rowley Paul
Published: December 2019

Combatting the spread of drug-resistant microbes requires new antifungal compounds with novel mechanisms of inhibition. Our lab investigates natural, proteinaceous toxins that are coded by double stranded RNA satellites found within Saccharomyces cerevisiae. Commonly known as “killer yeasts”, toxin-producing strains of S. cerevisiae have been found to inhibit the growth of many important fungal pathogens. To assess the value of killer yeasts for medical application we tested over 6,000 interactions between killer yeasts and various important fungal pathogens. To determine the susceptibility of fungi to killer toxins, we competed killer yeasts against a dilute lawn of a fungal pathogen; an active toxin produces a zone of growth inhibition in the lawn around the killer yeast. We determined that Candida glabratais broadly susceptible to killer toxins, while other Candida species showed little or no susceptibility. Of the 90 strains of S. cerevisiae tested against C. glabrata seven were capable of inhibiting all clinical strains of drug-resistant C. glabrata available from the Center for Disease Control and the U.S. Department of Agriculture. In our evaluation of these toxins as potential antifungal therapeutics we confirmed their inhibitory capability against C. glabrata under physiological conditions. Based on the results of these tests and prior knowledge about killer yeasts, we believe that these toxins show a potential as antifungal therapeutic precursors to treat recalcitrant infections caused by C. glabrata.
Niche-selective inhibition of pathogenic Th17 cells by targeting metabolic redundancy
Authors: Wu Lin , Hollinshead Kate E.R. , Hao Yuhan , Au Christy , Kroehling Lina , Ng Charles , Lin Woan-Yu , Li Dayi , Silva Hernandez Moura , Shin Jong , Lafaille Juan J. , Possemato Richard , Pacold Michael E. , Papagiannakopoulos Thales , Kimmelman Alec C. , Satija Rahul , Littman Dan R.
Published: December 2019

Summary

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.
CsHscB as a novel TLR2 agonist from carcinogenic liver fluke Clonorchis sinensis modulates host immune response
Authors: Yan Chao , Fan Fang , Zhang Yu-Zhao , Wu Jing , Dong Xin , Liu Hai-Liang , Fan Chun-Yang , Yu Qian , Wang Liang , Li Xiang-Yang , Wang Yu-Gang , Chen Jia-Xu , Tang Ren-Xian , Zheng Kui-Yang
Published: December 2019

Clonorchis sinensis-a fluke dwelling on the intrahepatic bile ducts causes clonorchiasis. During C. sinensis infection, worm-host interaction results in activation of PRRs and further triggers immune responses which determine the outcome of infection. However, the mechanisms by which pathogen-associated molecules patterns from C. sinensis interacted with TLRs were poorly understood. In the present study, we identified a ∼34 kDa lipoprotein CsHscB from C. sinensis which physically bound with TLR2. We also found that recombinant CsHscB (rCsHscB) potently activated macrophage to express various proteins including TLR2, CD80, MHCII, and cytokines like IL-6, TNF-α, and IL-10 in a TLR2-dependent manner but rCsHscB failed to induce IL-10 in macrophages from Tlr2-/- mice. Moreover, ERK1/2 activation was required for rCsHscB-induced IL-10 production in macrophages. In vivo study revealed that rCsHscB triggered a high induction of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. Our data thus demonstrate that rCsHscB from C. sinensis is an unidentified TLR2 agonist with immune regulatory activities, and may have some therapeutic implications in future beyond parasitology.
An upstream open reading frame represses translation of chicken PPARγ transcript variant 1
Authors: Chu Yankai , Huang Jiaxin , Ma Guangwei , Cui Tingting , Yan Xiaohong , Li Hui , Wang Ning
Published: December 2019

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipogenesis. The PPARγ gene produces various transcripts with different 5′-untranslated regions (5′ UTRs) because of alternative promoter usage and splicing. The 5′ UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5′ UTRs in the posttranscriptional regulation of PPARγ expression remain largely unclear. In this study, we investigated the effects of 5′ UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPARγ 5′ UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPARγ transcript 1 (PPARγ1) possessed an upstream open reading frame (uORF) in its 5′ UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPARγ protein expression, but decreased Renilla luciferase and PPARγ1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5′ UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPARγ1 mRNA. Collectively, our results indicate that the uORF represses the translation of chicken PPARγ1 mRNA.
Oxytocin has sex-specific effects on social behaviour and hypothalamic oxytocin immunoreactive cells but not hippocampal neurogenesis in adult rats
Authors: Duarte-Guterman Paula , Lieblich Stephanie E. , Qiu Wansu , Splinter Jared E.J. , Go Kimberly A. , Casanueva-Reimon Laura , Galea Liisa A.M.
Published: December 2019

Oxytocin regulates social behaviours, pair bonding and hippocampal neurogenesis but most studies have used adult males. Our study investigated the effects of oxytocin on social investigation and adult hippocampal neurogenesis in male and female rats. Oxytocin has poor penetration of the blood-brain barrier, therefore we tested a nanoparticle drug, TRIOZANTM (Ovensa Inc.), which permits greater blood-brain-barrier penetration. Adult male and female rats were injected daily (i.p.) for 10 days with either: oxytocin in PBS (0.5 or 1.0 mg/kg), oxytocin in TRIOZANTM (0.5 or 1.0 mg/kg), or vehicle (PBS) and tested for social investigation. Oxytocin decreased body mass and increased social investigation and number of oxytocin-immunoreactive cells in the supraoptic nucleus (SON) of the hypothalamus in male rats only. In both sexes, oxytocin decreased the number of immature neurons (doublecortin+ cells) in the ventral hippocampus and reduced plasma 17β-estradiol levels in a dose- and delivery-dependent way. Oxytocin in TRIOZANTM reduced sedation observed post-injection and increased some central effects (oxytocin levels in the hypothalamus and ventral hippocampus neurogenesis) relative to oxytocin in PBS indicating that the nanoparticle may be used as an alternative brain delivery system. We showed that oxytocin has sex-specific effects on social investigation, body mass, sedation, and the oxytocin system. In contrast, similar effects were observed in both sexes in neurogenesis and plasma 17β-estradiol. Our work suggests that sex differences in oxytocin regulation of brain endpoints is region-specific (hypothalamus versus hippocampus) and that oxytocin does not promote social investigation in females.
Cancer modeling in colorectal organoids reveals intrinsic differences between oncogenic RAS and BRAF variants
Authors: Post Jasmin B. , Hami Nizar , Lohuis Jeroen , van de Ven Marieke , de Korte-Grimmerink Renske , Stangl Christina , Stelloo Ellen , Verlaan Ingrid , van Rheenen Jacco , Snippert Hugo J.G.
Published: December 2019

Colorectal cancers (CRCs) with oncogenic mutations in RAS and BRAF are associated with anti-EGFR therapy resistance. Consequently, all RAS mutant CRC patients are being excluded from this therapy. However, heterogeneity in drug response has been reported between RAS mutant CRC patients. It is poorly understood to what extent such differences are derived from different genetic backgrounds or intrinsic differences between the various RAS pathway mutations. Therefore, using CRISPR technology we generated an isogenic panel of patient-derived CRC organoids with various RAS pathway mutations (i.e. KRASG12D, BRAFV600E, KRASG13D and NRASG12D). All RAS pathway mutants promote ERK activation and tumor growth. However, KRASG12D and BRAFV600E mutations in particular conferred robust resistance to anti-EGFR therapy, both in vitro and in vivo. Moreover, untreated KRASG13D mutants showed fastest growth in mice but remained sensitive to anti-EGFR therapy. Together, introducing mutation-specific oncogene signaling in CRC organoids resembles clinical phenotypes and improves understanding of genotype-phenotype correlations.
Transforming growth factor-beta signaling via ALK1 and ALK5 regulates distinct functional pathways in vein graft intimal hyperplasia
Authors: Low EL , Schwartze JT , Kurkiewicz A , Pek M , Kelly DJ , Shaw AS , Thorikay M , McClure J , McBride M , Arias-Rivas S , Francis SE , Morrell NW , Delles C , Herzyk P , Havenga MJ , Nicklin SA , Ten Dijke P , Baker AH , Bradshaw AC
Published: December 2019

Rationale

Transforming growth factor-beta (TGFβ) is tightly regulated at multiple levels, with regulation at the receptor level now recognized as a key determinant of the cellular response to this pleiotropic cytokine. TGFβ promotes saphenous vein graft neointima formation after coronary artery bypass graft (CABG) surgery, inducing smooth muscle cell (SMC) hyperplasia and fibrosis by signaling via activin receptor-like kinase 5(ALK5). However, the role of the alternate TGFβ receptor ALK1 remains completely unknown.

Objective

To define the receptor pathways activated by TGFβ in SMCs and their mechanistic importance during CABG neointima formation.

Methods and results

Radioligand co-IP assays revealed direct interactions between TGFβ, ALK5 and ALK1 in primary saphenous vein graft SMC (HSVSMC) from patients undergoing CABG. Knockdown and pharmacological inhibition of ALK5 or ALK1 in HSVSMC significantly attenuated TGFβ-induced phosphorylation of receptor-regulated (R)-Smads 2/3 and 1/5, respectively. Microarray profiling followed by qRT-PCR validation showed that TGFβ induced distinct transcriptional networks downstream of ALK5 or ALK1, associated with HSVSMC contractility and migration, respectively and confirmed using migration assays as well as qRT-PCR and western blot assays of contractile SMC markers. scRNAseq analysis of TGFβ-treated HSVSMC identified distinct subgroups of cells showing ALK5 or ALK1 transcriptional responses, while RNA velocity analyses indicated divergence in differentiation towards ALK5 or ALK1-dominant lineages. ALK1, ALK5 and their downstream effectors pSmad1/5 and pSmad2/3 were localized to αSMA+ neointimal SMCs in remodelled mouse vein grafts. Pharmacological inhibition or genetic ablation of Smad1/5 substantially reducing neointima formation following acute vascular injury. Notably, expression and activation of ALK1, ALK5 and their respective downstream R-Smads was already evident in hyperplastic saphenous veins prior to grafting.

Conclusions

Whilst canonical TGFβ signaling via ALK5 promotes a contractile HSVSMC phenotype, transactivation of ALK1 by TGFβ induces neointima formation by driving cell migration. Restoring the balance between ALK1 and ALK5 in HSVSMC may represent a novel therapeutic strategy for vein graft failure.
Assessing Environmental RNAi in a Non-Model Organism
Authors: Mondal Mosharrof , Peter Jacob , Scarbrough Obrie , Flynt Alex
Published: December 2019

RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation in invertebrates and has applications that includes control of pests. Successful RNAi technologies are dependent on delivery method. The most convenient method is likely feeding which is effective in some animals while others are insensitive. Thus, there is a need to develop RNAi technology on a per-species basis, which will require a comprehensive approach for assessing small RNA production from synthetic nucleic acids.

Using a biochemical and sequencing approaches we investigated the metabolism of ingested RNAs using the two-spotted spider mite, Tetranychus urticae, as a model for RNAi insensitivity. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. To identify RNAi substrates in T. urticae we characterized processing of synthetic RNAs and those derived from plant transcripts ingested during feeding. Through characterization of read size length and overlaps of small RNA reads, visualization methods were developed that facilitate distinguish trans-acting small RNAs from degradation fragments.

Using a strategy that delineates small RNA classes, we found a variety of RNA species are gated into spider mite RNAi pathways, however, potential mature trans-acting RNAs appear very unstable and rare. This suggests spider mite RNAi pathway products that originate as ingested materials may be preferentially metabolized instead of converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers.
Genetic diversity and population structure of the endangered endemic speciesPaeonia decompositafrom China and implications for its conservation
Authors: Wang Shi-Quan
Published: December 2019

Paeonia decomposita, endemic to China, has important ornamental, medicinal and economic value and is regarded as a threatened endangered plant. The genetic diversity and structure have seldom been described. A conservation management plan is not currently available. In present study, 16 pairs of SSR primers were used to evaluate genetic diversity and population structure. A total of 122 alleles were obtained with a mean of 7.625 alleles per locus. The expected heterozygosity (He) varied from 0.043 to 0.901 (mean 0.492). Moderate genetic diversity (He=0.405) among populations were revealed, with Danba identified as the center of genetic diversity. Mantel tests revealed a significant positive correlation between geographic and genetic distance among populations (r=0.592, P=0.0001), demonstrating consistency with the isolation by distance model. Analysis of molecular variance (AMOVA) results indicated that the principal genetic variation existed within populations (73.48%) rather than among populations (26.52%). Bayesian structure analysis and principal coordinate analysis (PCoA) supported classification of the populations into three clusters. Based on the level of observed genetic diversity, three management unints were proposed as conservation measures. The results will be beneficial for the conservation and exploitation of the species, providing a theoretical basis for further research on its evolution and phylogeography.

Hightlights

Genetic diversity among populations was moderate inPaeonia decomposita

There is significant positive correlation between geographic and genetic distance among populations, consistent with the isolation by distance model

Principal genetic variation existed within populations rather than among populations.

The populations divided into three clusters.

Three management unints were proposed as conservation measures.