bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitativein vivoanalyses reveal a complex pharmacogenomic landscape in lung adenocarcinoma
Authors: Li Chuan , Lin Wen-Yang , Rizvi Hira , Cai Hongchen , McFarland Christopher D. , Rogers Zoë N. , Yousefi Maryam , Winters Ian P. , Rudin Charles M. , Petrov Dmitri A. , Winslow Monte M.
Published: January 2020

The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most important gaps in enabling the effective use of cancer therapies. Here, we couple a multiplexed and quantitative platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responsesin vivo. We uncover a surprisingly complex map of genotype-specific therapeutic responses, with over 20% of possible interactions showing significant resistance or sensitivity. We validate one of these interactions - the resistance of Keap1 mutant tumors to platinum therapy - using a large patient response dataset. Our results highlight the importance of understanding the genetic determinants of treatment responses in the development of precision therapies and define a strategy to identify such determinants.
Acoustic Ejection Mass Spectrometry for High-Throughput Analysis
Authors: Zhang Hui , Liu Chang , Hua Wenyi , Ghislain Lucien P. , Liu Jianhua , Aschenbrenner Lisa , Noell Stephen , Dirico Kenneth , Lanyon Lorraine F. , Steppan Claire M. , Arnold Don W. , Covey Thomas R. , Datwani Sammy S. , Troutman Matthew D.
Published: January 2020

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI) MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision and accuracy; subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro and in vivo adsorption, distribution, metabolism, elimination, pharmacokinetic (PK) and biomarker analysis, and HT parallel medicinal chemistry.

One Sentence Summary

ADE-OPI-MS is a transformational analytical platform that increases mass spectrometry utility via sub-second speed and non-contact sampling.
Incidence and Risk Factors of Second Primary Cancer after the Initial Primary Human Papillomavirus Related Neoplasms
Authors: Shen Jiayi , Zhou Huaqiang , Liu Jiaqing , Zhang Zhonghan , Fang Wenfeng , Yang Yunpeng , Hong Shaodong , Ma Yuxiang , Zhou Ting , Zhang Yaxiong , Zhao Hongyun , Huang Yan , Zhang Li
Published: January 2020

Background

Human papillomavirus (HPV)-related cancers are nowadays associated with better survival. However, comprehensive studies in second primary cancer (SPC) after the initial primary HPV-related cancer still remain warranted. Therefore, this study was designed to analyse the incidence and risk factors of SPC after HPV-related cancer.

Methods

We identified 86,790 patients diagnosed with initial primary HPV-related cancer between 1973 and 2010 in the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) 9 database. Standardized incidence ratio (SIR) and cumulative incidence were calculated to assess the risk of SPC after HPV-related cancer. Subdistribution hazard regression was also conducted to figure out the risk factors.

Results

SIR of SPC after HPV-related cancer was 1.60 (95% confidence interval [CI] 1.55-1.65) for male and 1.25 (95% CI 1.22-1.28) for female. SIR of second primary HPV-related cancer (7.39 [95% CI 6.26-8.68] male and 4.35 [95% CI 4.04-4.67] female) was obviously higher than that of second primary HPV-unrelated cancer (1.54 [95% CI 1.49-1.60] male and 1.16 [95% CI 1.13-1.19] female). 5-year cumulative incidence of SPC was 7.22% (95% CI 6.89%-7.55%) for male and 3.72% (95% CI 3.58%-3.88%) for female. Risk factors of SPC included being married and initial primary cancer (IPC) diagnosed at earlier stage for both gender, and IPC diagnosed at older age as well as surgery performed for female only.

Conclusion

Patients diagnosed with HPV-related cancer are more likely to develop another primary cancer, as compared with the age-specific reference population. Patients with the risk factors claimed in this study are suggested to screen for SPC regularly. According to the elevation of SIR of HPV-related SPC, it is suggested that part of the HPV-related SPC cases may be caused by the persistent infection of HPV.
Genomic determination of relative risks for Clostridioides difficile infection from asymptomatic carriage in ICU patients
Authors: Worley Jay , Delaney Mary L. , Cummins Christopher K. , DuBois Andrea , Klompas Michael , Bry Lynn
Published: January 2020

Background

Clostridioides difficile infections (CDIs) are among the most prevalent hospital-associated infections (HAIs), particularly for intensive care unit (ICU) patients. The risks for developing active CDI from asymptomatic carriage of C. difficile are not well understood.

Methods

We identified asymptomatic C. difficile carriage among 1897 ICU patients, using rectal swabs from an existing ICU vancomycin-resistant Enterococci (VRE) surveillance program. C. difficile isolates from VRE swabs, and from C. difficile-positive stool samples, were genome sequenced to assess clonal relationships among isolates from asymptomatic carriers and CDI patients. Integrated genomic and epidemiologic analyses identified multiple cases of asymptomatic carriers who developed CDI, and of asymptomatic transmission of C. difficile to naïve patients.

Results

Genomic analyses identified diverse strains in infected patients and asymptomatic carriers. 7.4% of ICU patients asymptomatically carried C. difficile. 69% of isolates carried an intact toxin locus. In contrast, 96% of C. difficile stool isolates were toxigenic. CDI rates in asymptomatic carriers of toxigenic strains were 5.3%, versus 0.57% in non-carriers. The relative risk for CDI with asymptomatic carriage of a toxigenic strain was 9.32 (95% CI=3.25-26.7). Genomic identification of clonal clusters supported epidemiologic analyses for asymptomatic transmission events, with spatial-temporal overlaps identified in 13 of 28 cases.

Conclusions

Our studies provide the first genomically-confirmed assessments of CDI relative risk from asymptomatic carriage of toxigenic strains and highlight the complex dynamics of asymptomatic transmission in ICUs. C. difficile screening can be implemented within existing HAI surveillance programs and, with isolation of asymptomatic carriers, has potential to reduce these risks.

Relative risks for C. difficile infections rise to 9.32 in asymptomatic ICU patients carrying toxigenic strains. Integrated genomic and epidemiologic analyses illustrate functional use of C. difficile genomic data to identify asymptomatic transmission events and assist in outbreak investigations.
Spatio-temporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis
Authors: Marmor-Kollet Hagai , Siany Aviad , Kedersha Nancy , Knafo Naama , Rivkin Natalia , Danino Yehuda M. , Olender Tsviya , Cohen Nir , Moens Thomas , Higginbottom Adrian , Knock John Cooper- , Eitan Chen , Cohen Beata Toth , Van Den Bosch Ludo , Anderson Paul , Ivanov Pavel , Geiger Tamar , Hornstein Eran
Published: January 2020

Cytoplasmic stress granules (SG) form in response to a variety of cellular stresses by phase-separation of proteins associated with non-translating mRNAs. SG provide insight into the biology of neurodegeneration, including amyotrophic lateral sclerosis (ALS) because they approximate some of the molecular conditions for nucleation of insoluble aggregates in neuropathological inclusions. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SG undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of SG temporal disassembly sequence, using multi-bait APEX proximity-proteomics. We discover 109 novel SG-proteins and characterize at proteomic resolution two biophysically distinct SG substructures. We further demonstrate that dozens of additional proteins are recruited to SG specifically during disassembly, indicating that it is a highly regulated process. The involved proteins link SG disassembly, to mitochondrial biology and the cytoskeleton. Parallel analysis with C9ORF72-associated dipeptides, which are found in patients with ALS and frontotemporal dementia, demonstrated compositional changes in SG during the course of disassembly and focused our attention on the roles SUMOylation in SG disassembly. We demonstrate that broad SUMOylation of SG-proteins is required for SG disassembly and is impaired by C9ORF72-associated dipeptides, representing an unexplored potential molecular mechanism of neurodegeneration. Altogether, out study fundamentally increases the knowledge about SG composition in human cells by dissecting the SG spatio-temporal proteomic landscape, provides an in-depth resource for future work on SG function and reveals basic and disease-relevant mechanisms of SG disassembly.

Highlights

Multi bait APEX proximity labelling reveals 109 novel SG proteins and two distinct SG substructures.

Proteomic dissection of SG temporal disassembly under basal conditions and with a model of neurodegeneration.

Disassembly-engaged proteins (DEPs) include SUMO ligases that are recruited during normal disassembly and dysregulated in ALS-like conditions.

Pervasive SG protein SUMOylation during SG disassembly is impaired by ALS-like conditions.

Abstract Figure
Cyclin E1 protein is stabilized inBRCA1mutated breast cancers leading to synergy between CDK2 and PARP inhibitors
Authors: Aziz Diar , Portman Neil , Fernandez Kristine J. , Lee Christine , Alexandrou Sarah , Llop-Guevara Alba , Yong Aliza , Wilkinson Ashleigh , Sergio C. Marcelo , Ferraro Danielle , Etemadmoghadam Dariush , Bowtell David , Investigators kConFab , Serra Violeta , Waring Paul , Lim Elgene , Caldon C. Elizabeth
Published: January 2020

Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly to targeted therapies and chemotherapies. In order to define therapeutically targetable subsets of BLBC we examined two markers: cyclin E1 andBRCA1loss. In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, and define therapeutic subsets. We tested the same hypothesis for BLBC.

Using a BLBC cohort enriched forBRCA1loss, we identified convergence betweenBRCA1loss and high cyclin E1 expression, in contrast to HGSOC in whichCCNE1amplification drives increased cyclin E1 gene expression. Instead,BRCA1loss stabilized cyclin E1 during the cell cycle. Using siRNA we showed thatBRCA1loss leads to stabilization of cyclin E1 by reducing phospho-cyclin E1-T62, and conversely the overexpression ofBRCA1increased phospho-T62. Mutation of cyclin E1-T62 to alanine increased cyclin E1 stability. We showed that tumors with high cyclin E1/BRCA1mutation in the BLBC cohort had decreased phospho-T62, supporting this hypothesis.

Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated inBRCA1mutant cancers, we hypothesized that CDK2 inhibition would sensitize these cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized with PARP inhibitors to reduce cell viability inBRCA1mutated cell lines. Treatment of BLBC patient-derived xenograft models with combination PARP and CDK2 inhibition led to tumor regression and increased survival. We conclude thatBRCA1status and high cyclin E1 have potential as predictive biomarkers to dictate the therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC.
Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome
Authors: Tosar Juan Pablo , Segovia Mercedes , Gámbaro Fabiana , Akiyama Yasutoshi , Fagúndez Pablo , Costa Bruno , Possi Tania , Hill Marcelo , Ivanov Pavel , Cayota Alfonso
Published: January 2020

A major proportion of extracellular RNAs (exRNAs) do not co-isolate with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed RI-SEC-seq: a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes can be sensed by dendritic cells. Extracellular ribosomes and/or tRNAs could therefore be decoded as damage-associated molecular patterns.
Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program
Authors: Srinivasan Rajalakshmi , Walvekar Adhish S. , Seshasayee Aswin , Laxman Sunil
Published: January 2020

Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells must manage biomolecule supply to balance constructive metabolism with high translation, through coordinated gene expression programs. Global growth programs couple increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis, and how this is collectively managed is a fundamental question. Conventionally, the role of the Gcn4/ATF4 transcription factor has been studied only in the context of amino acid starvation. However, high Gcn4/ATF4 has been observed in contexts of rapid cell proliferation, and the specific role of Gcn4 in growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum through which metabolic supply dependent sustenance of translation outputs is maintained. Integrating time-matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lys/arg enriched transcripts, which include the translation machinery. The sustained Gcn4 dependent lys/arg supply is required to maintain sufficient translation capacity, by allowing the synthesis of the translation machinery itself. Gcn4 thereby enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controled using the same Gcn4 transcriptional outputs, in entirely distinct contexts.
Learning to Integrate an Artificial Sensory Device: Early Bayesian Integration and Conscious Perception
Authors: Mahani Mohammad-Ali Nikouei , Bausenhart Karin Maria , Ulrich Rolf , Ahmadabadi Majid Nili
Published: January 2020

The present study examines how artificial tactile stimulation from a novel non-invasive sensory device is learned and integrated with information from another sensory system. Participants were trained to identify the direction of visual dot motion stimuli with a low, medium, and high signal-to-noise ratio. In bimodal trials, this visual direction information was paired with reliable symbolic tactile information. Over several training blocks, discrimination performance in unimodal tactile test trials and subjects’ confidence in their decision improved, indicating that participants were able to associate the visual and tactile information consciously and thus learned the meaning of the symbolic tactile cues. Formal analysis of the results in bimodal trials showed that both modalities are being integrated already in the early learning phases. Our modeling results revealed that this integration is consistent with a Bayesian model, which is an optimal integration of sensory information. Furthermore, we showed that a confidence-based Bayesian integration explains the observed behavioral data better than the classical variance-based Bayesian integration. Thus, the present study demonstrates that humans can consciously learn and integrate an artificial sensory device that delivers symbolic tactile information. This finding connects the field of multisensory integration research to the development of sensory substitution systems.
Dynamic landscape of protein occupancy across the Escherichia coli chromosome
Authors: Freddolino Peter L. , Amemiya Haley M. , Goss Thomas J. , Tavazoie Saeed
Published: January 2020

Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (IPOD-HR), and present a pilot application to E. coli. We observe that the E. coli protein-DNA interactome organizes into two distinct prototypic features: (1) highly dynamic condition-dependent transcription factor occupancy, and (2) robust kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence-specificity models for the majority of active transcription factors. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria which are not amenable to routine genetic manipulation.
Deoxyribozyme-based Method for Site-specific Absolute Quantification of N6-methyladenosine Modification Fraction
Authors: Bujnowska Magda , Zhang Jiacheng , Dai Qing , Heideman Emily M. , Fei Jingyi
Published: January 2020

N6-methyladenosine (m6A) is the most prevalent modified base in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRNA). Although candidate sites for m6A modification are identified at the transcriptomic level, site-specific quantification methods for m6A modifications are still limited. Herein, we present a facile method implementing deoxyribozyme that preferentially cleaves the unmodified RNA. We leverage reverse transcription and real-time quantitative PCR along with key control experiments to quantify the absolute methylation fraction of specific m6A sites. We validate the accuracy of the method using synthetic RNA with controlled methylation fraction and apply our method on several endogenous sites that were previously identified in sequencing-based studies. This method provides a time and cost-effective approach for absolute quantification of the m6A fraction at specific loci, expanding the current toolkit for studying RNA modifications.
Factorial study of the RNA-seq computational workflow identifies biases as technical gene signatures
Authors: Simoneau Joël , Gosselin Ryan , Scott Michelle S.
Published: January 2020

RNA-seq is a modular experimental and computational approach that aims in identifying and quantifying RNA molecules. The modularity of the RNA-seq technology enables adaptation of the protocol to develop new ways to explore RNA biology, but this modularity also brings forth the importance of methodological thoroughness. Liberty of approach comes with the responsibility of choices, and such choices must be informed. Here, we present an approach that identifies gene group specific quantification biases in currently used RNA-seq software and references by processing sequenced datasets using a wide variety of RNA-seq computational pipelined, and by decomposing these expression datasets using an independent component analysis matrix factorisation method. By exploring the RNA-seq pipeline using a systemic approach, we highlight the yet inadequately characterized central importance of genome annotations in quantification results. We also show that the different choices in RNA-seq methodology are not independent, through interactions between genome annotations and quantification software. Genes were mainly found to be affected by differences in their sequence, by overlapping genes and genes with similar sequence. Our approach offers an explanation for the observed biases by identifying the common features used differently by the software and references, therefore providing leads for the betterment of RNA-seq methodology.
Grip strength is associated with retinal and choroidal thickness in type 2 diabetes mellitus patients without retinopathy
Authors: Qiu Zihan , Wang Wei , Tan Yan , He Miao , Wang Lanhua , Li Yuting , Gong Xia , Huang Wenyong
Published: February 2020

Objective

To determine the relationship between grip strength and retinal or choroidal thickness in Chinese patients with type 2 diabetes mellitus.

Design

Observational study-cross-sectional design.

Setting and Participants

The study was conducted among diabetes patients without retinopathy registered in the community health system in Guangzhou, China.

Measures

Grip strength was measured twice for each hand with a dynamometer in kilograms (kg). The retinal and choroidal thickness in macular Early Treatment Diabetic Retinopathy Study (ETDRS) sectors were measured by commercial swept-source optical coherence tomography (SS-OCT; DRI OCT-2 Triton; Topcon, Tokyo, Japan).

Results

A total of 1,029 patients were included in the study. Both retinal thickness and choroidal thickness increased with the higher quartile of grip strength. Regression analyses indicated that the average retinal and choroidal thickness increased by 0.14 μm (95%CI: 0.03-0.25 μm, P=0.011) and 0.57 μm (95%CI: 0.03-1.11 μm, P=0.037) for each additional kg of grip strength following adjustment for age and gender. Further adjustments were made for axial length, HbA1c, length of time the patient had diabetes, insulin usage, height, weight and systolic and diastolic blood pressure, which resulted in an average retinal and choroidal thickness increase of 0.13 μm (95%CI: 0.02-0.24 μm, P=0.024) and 0.65 μm (95%CI: 0.13-1.16 μm, P=0.013) for each additional kg of grip strength. Consistent results were obtained in the analyses in ETDRS 9 sectors.

Conclusion

Greater hand grip strength was found to be significantly associated with thicker retinal and choroidal layers in diabetic patients. Grip strength may provide a useful indicator of retinal health in diabetic patients. Further studies are needed to determine directionality and causality, and to examine whether improving muscle strength has a positive effect on retinal and choroidal thickness.

Article summary

Strengths and limitations of this study

Grip strength is an indicator of upper limb muscle function and tension, and it is one of the important indicators of aging in the human population. This is one of the few studies to examined the relationship between grip strength and retinal thickness and choroidal thickness in a large sample size of patients with diabetes mellitus.

Greater hand grip strength was found to be significantly associated with thicker retinal and choroidal thickness in diabetic patients, and grip strength may provide an easily-administered marker of retinal health in diabetic patients.

Causal inferences could not be inferred due to the cross-sectional design of the study.

The dynamic changes in grip strength and their impact on retinal and choroidal thickness are warranted to be explored in longitudinal studies.
LAG3 is a Central Regulator of NK Cell Cytokine Production
Authors: Narayanan Sriram , Ahl Patricia J. , Bijin Veonice Au , Kaliaperumal Nivashini , Lim Seng Gee , Wang Cheng-I , Fairhurst Anna-Marie , Connolly John E.
Published: February 2020

Natural killer (NK) cells are innate effectors, which play a crucial role in controlling viral infections. Administration of IFN-α has shown promising results as a therapeutic, controlling HIV, and chronic viral hepatitis. However the downstream mechanisms by which IFN-α mediates its anti-viral effects is largely unknown. In this investigation, we evaluated the impact of IFN-α on peripheral blood NK cells from healthy donors. High dimensional flow cytometry analysis of NK cell surface receptors following exposure to IFN-α showed an increased expression of the check point inhibitor LAG3. Further characterization revealed that LAG3 was expressed in a subset of NK cells with high expression of activation and maturation markers. Assessment of metabolic pathways showed that LAG3+ NK cells had enhanced rates of glycolysis and glycolytic capacity, suggesting that it is a primed effector subset with enhanced glucose metabolism. Inhibition of LAG3 on NK cells using antibodyin vitroresulted in a profound increase in secretion of cytokines IFN-γ, TNF-α, MIP-1α and MIP-1β, without affecting the cytotoxic activity. Taken together, these results showed that LAG3 is a negative regulator of cytokine production by mature NK cells.
The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation
Authors: Rashad Sherif , Han Xiaobo , Sato Kanako , Mishima Eikan , Abe Takaaki , Tominaga Teiji , Niizuma Kuniyasu
Published: February 2020

tiRNAs are small non-coding RNAs produced when tRNA is cleaved under stress. tRNA methylation modifications has emerged in recent years as important regulators for tRNA structural stability and sensitivity to cleavage and tiRNA generation during stress, however, the specificity and higher regulation of such a process is not fully understood. Alkbh1 is a m1A demethylase that leads to destabilization of tRNA and enhanced tRNA cleavage. We examined the impact of Alkbh1 targeting via gene knockdown or overexpression on B35 rat neuroblastoma cell line fate following stresses and on tRNA cleavage. We show that Alkbh1 impact on cell fate and tRNA cleavage is a stress specific process that is impacted by the demethylating capacity of the cellular stress in question. We also show that not all tRNAs are cleaved equally following Alkbh1 manipulation and stress, and that Alkbh1 KD fails to rescue tRNAs from cleavage following demethylating stresses. These findings shed a light on the specificity and higher regulation of tRNA cleavage and should act as a guide for future work exploring the utility of Alkbh1 as a therapeutic target for cancers or ischemic insult.
Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems
Authors: Yu Timothy C. , Liu Winnie L. , Brinck Marcia , Davis Jessica E. , Shek Jeremy , Bower Grace , Einav Tal , Insigne Kimberly D. , Phillips Rob , Kosuri Sriram , Urtecho Guillaume
Published: February 2020

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case ofEscherichia coliinducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8,269 rationally designed IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. Using these data, we fit a statistical mechanics model that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three novel promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.
Effects of host species identity and diet on biodiversity of oral and rectal microbiomes of Puerto Rican bats
Authors: Presley Steven J. , Graf Joerg , Hassan Ahmad F. , Sjodin Anna R. , Willig Michael R.
Published: February 2020

Microbiomes perform vital functions for their mammalian hosts, making them potential drivers of host evolution. Understanding effects of environmental factors and host characteristics on the composition and biodiversity of microbiomes may provide novel insights into the origin and maintenance of these symbiotic relationships. Our goals were to (1) characterize biodiversity of oral and rectal microbiomes of bats in Puerto Rico; and (2) determine the effects of geographic location and host characteristics on that biodiversity. We collected bats and their microbiomes from 3 sites, and used 4 metrics (species richness, Shannon diversity, Camargo evenness, Berger-Parker dominance) to characterize biodiversity. We evaluated the relative importance of site, host sex, host species identity, and host foraging guild on microbiome biodiversity. Microbiome biodiversity was highly variable among conspecifics. Geographical location exhibited consistent effects, whereas host sex did not do so. Within each host guild, host species exhibited consistent differences in oral and rectal microbiome biodiversity. Oral microbiome biodiversity was indistinguishable between guilds, whereas rectal microbiome biodiversity was significantly greater in carnivores than in herbivores. The high intraspecific and spatial variation in microbiome biodiversity necessitate a large number of samples to isolate the effects of environmental or host characteristics on microbiomes. Species-specific biodiversity of oral microbiomes suggests these communities are structured by direct interactions with the host immune system via epithelial receptors. In contrast, the number of microbial taxa that a host gut supports may be contingent on the number and kinds of functions a host requires of its microbiome.
Covalent Protein Painting Reveals Structural Changes in the Proteome in Alzheimer Disease
Authors: Bamberger Casimir , Pankow Sandra , Martínez-Bartolomé Salvador , Ma Michelle , Diedrich Jolene , Rissman Robert A. , Yates John R.
Published: February 2020

The 3D structures of aberrant protein folds have been visualized in exquisite detail, yet no method has been able to quantitatively measure protein misfolding across a proteome. Here, we present Covalent Protein Painting (CPP), a mass spectrometry-based structural proteomics approach to quantify the accessibility of lysine ε-amines for chemical modification at the surface of natively folded proteins. We used CPP to survey 2,645 lysine residues in the proteome of HEK293T cellsin vivoand found that mild heat shock increased rather than decreased lysine accessibility for chemical modification. CPP was able to differentiate patients with Alzheimer disease (AD) or Lewy body disease (LBD) or both from controls based on relative accessibility of lysine residues K147, K137, and K28 in Tubulin-β, Succinate dehydrogenase, and amyloid-β peptide, respectively. The alterations of Tubulin-β and Succinate dehydrogenase hint to broader perturbations of the proteome in AD beyond amyloid-β and hyper-phosphorylated tau.
Structural and functional evaluation ofde novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens
Authors: Antanasijevic Aleksandar , Ueda George , Brouwer Philip JM , Copps Jeffrey , Huang Deli , Allen Joel D , Cottrell Christopher A , Yasmeen Anila , Sewall Leigh M , Bontjer Ilja , Ketas Thomas J , Turner Hannah L , Berndsen Zachary T , Klasse Per Johan , Crispin Max , Nemazee David , Moore John P , Sanders Rogier W , King Neil P , Baker David , Ward Andrew B
Published: February 2020

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP trimers.

Author Summary

Protein constructs based on soluble ectodomains of HIV glycoprotein (Env) trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library ofde novodesigned, protein nanoparticles of different geometries for their ability to present trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessedin vitroandin vivo. In addition to introducing a novel set of reagents for multivalent display of Env trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.
Evolution of sperm morphology in a crustacean genus with fertilization inside an open brood pouch
Authors: Duneau David , Möst Markus , Ebert Dieter
Published: February 2020

Abstract

Sperm is the most fundamental male reproductive feature. It serves the fertilization of eggs and evolves under sexual selection. Two components of sperm are of particular interest, their number and their morphology. Mode of fertilization is believed to be a key determinant of sperm length across the animal kingdom. External fertilization, unlike internal, favors small and numerous sperm, since sperm density is thinned out in the environment. Here, we study the evolution of sperm morphology in the genusDaphnia, where fertilization occurs in a receptacle, the brood pouch, where sperm can constantly be flushed out by a water current. Based on microscopic observations of sperm morphologies mapped on a phylogeny with 15Daphniaand 2 outgroup species, we found that despite the internal fertilization mode,Daphniahave among the smallest sperm recorded, as would be expected with external fertilization. Despite being all relatively small compared to other arthropods, sperm length diverged at least twice, once within each of theDaphniasubgeneraCtenodaphniaandDaphnia.Furthermore, species in the latter subgenus also lost the ability of cell compaction by extracellular encapsulation and have very polymorphic sperm with long, and often numerous, filopodia. We discuss the different strategies thatDaphniaevolved to achieve fertilization success in the females’ brood pouch.
Microbial community assembly in a multi-layer dendritic metacommunity
Authors: Wisnoski Nathan I. , Lennon Jay T.
Published: February 2020

A major goal of metacommunity ecology is to infer the local- and regional-scale processes that underlie community assembly. In dendritic ecological networks (e.g., stream metacommunities), branching and directional dispersal connectivity can alter the balance between local and regional factors during assembly. However, the implications of vertical habitat structure (e.g., planktonic versus benthic sediments) in dendritic metacommunities remain unclear. In this study, we analyzed the bacterial metacommunity of a fifth-order mountain stream network to assess habitat differences in the (1) dominant community assembly processes, (2) spatial scaling of community assembly processes, and (3) longitudinal variation in community assembly. Using taxonomic and phylogenetic null modeling approaches, we found habitat-specific spatial patterns of community assembly across the dendritic network. Compositional differences between planktonic and benthic communities were maintained by divergent species sorting, but stochasticity influenced assembly at local scales. Planktonic communities showed scale-dependent assembly, transitioning from convergent sorting at local scales to divergent sorting at regional scales, while sediment community assembly was less scale dependent (convergent sorting remained important across all scales). While divergent sorting structured headwaters in both habitat types, sediment communities converged in structure downstream. Taken together, our results show that vertical habitat structure regulates the scale-dependent processes of community assembly across the dendritic metacommunity.
Metagenomic Insights into Microbial Metabolisms of a Sulfur-Influenced Glacial Ecosystem
Authors: Trivedi Christopher B. , Stamps Blake W. , Lau Graham E. , Grasby Stephen E. , Templeton Alexis S. , Spear John R.
Published: February 2020

Biological sulfur cycling in polar, low-temperature ecosystems is an understudied phenomenon in part due to difficulty of access and the ephemeral nature of such environments. One such environment where sulfur cycling plays an important role in microbial metabolisms is located at Borup Fiord Pass (BFP) in the Canadian High Arctic. Here, transient springs emerge from the toe of a glacier creating a large proglacial aufeis (spring-derived ices) that are often covered in bright yellow/white sulfur, sulfate, and carbonate mineral precipitates that are accompanied by a strong odor of hydrogen sulfide. Metagenomic sequencing from multiple sample types at sites across the BFP glacial system produced 31 highly complete metagenome assembled genomes (MAGs) that were queried for sulfur-, nitrogen- and carbon-cycling/metabolism genes. Sulfur cycling, especially within the Sox complex of enzymes, was widespread across the isolated MAGs and taxonomically associated with the bacterial classesAlpha-, Beta-, Gamma-, andEpsilon- Proteobacteria. While this does agree with previous research from BFP implicating organisms within theGamma-andEpsilon- Proteobacteriaas the primary classes responsible for sulfur oxidation, our new data suggests putative sulfur oxidation by organisms withinAlpha-andBeta- Proteobacterialclasses which was not predicted. These findings indicate that in a low-temperature, ephemeral sulfur-based environment such as this, functional redundancy may be a key mechanism that microorganisms use to co-exist whenever energy is limited and/or focused by redox chemistry.

Importance

Borup Fiord Pass is a unique environment characterized by a sulfur-enriched glacial ecosystem, in the low-temperature environment of the Canadian High Arctic. This unique combination makes BFP one of the best analog sites for studying icy, sulfur-rich worlds outside of our own, such as Europa and Mars. The site also allows investigation of sulfur-based microbial metabolisms in cold environments here on Earth. Herein, we report whole genome sequencing data that suggests sulfur cycling metabolisms at BFP are more widely used across bacterial taxa than predicted. From our data, the metabolic capability of sulfur oxidation among multiple community members appears likely due to functional redundancy within their genomes. Functional redundancy, with respect to sulfur-oxidation at BFP, may indicate that this dynamic ecosystem hosts microorganisms that are able to use multiple sulfur electron donors alongside other important metabolic pathways, including those for carbon and nitrogen.
The branched chain aminotransferase IlvE promotes growth, stress resistance and pathogenesis ofListeria monocytogenes
Authors: Passalacqua Karla D. , Zhou Tianhui , Washington Tracy A. , Abuaita Basel H. , Sonenshein Abraham L. , O’Riordan Mary X.D.
Published: February 2020

The bacterial plasma membrane is a key interface during pathogen-host interactions, and membrane composition enhances resistance against host antimicrobial defenses. Branched chain fatty acids (BCFAs) are the major plasma membrane component in the intracellular Gram-positive pathogenListeria monocytogenes(Lm) and BCFA metabolism is essential for Lm growth and virulence. BCFA synthesis requires branched chain amino acids (BCAAs), and the BCAA Isoleucine (Ile) is a necessary substrate for the predominant membrane anteiso-BCFAs (ai-BCFAs) as well as an environmental signal for virulence regulation in Lm. In this study, we explored how two proteins that metabolize or sense Ile contribute to Lm growth, BCFA metabolism, and virulence. The IlvE aminotransferase incorporates Ile into ai-BCFAs, while CodY is an Ile-sensing regulator that coordinates BCAA synthesis and virulence gene expression. Analysis of deletion mutants lacking IlvE (ΔilvE) or CodY (ΔcodY) revealed a major role for IlvE under nutrient restriction and stress conditions. Cultures of the ΔilvEmutant contained proportionally less ai-BCFAs relative to wild type, while of the ΔcodYmutant had a lower proportion of ai-BCFAs in stationary phase, despite containing more cell-associated Ile. Both ΔilvEand ΔcodYmutants required exogenous Ile for optimal growth, but the ΔilvEmutant had an absolute requirement for Valine and Leucine when Ile was absent. IlvE was also necessary for resistance to membrane stress, cell-to-cell spread, infection of primary macrophages, and virulence in mice. Our findings implicate IlvE as an integral aspect of Lm stress resistance and emphasize the central importance of Ile in Lm growth and virulence.
An allometric scaling approach to estimate epiphytic bryophyte biomass in tropical montane cloud forests
Authors: Lai Guan-Yu , Liu Hung-Chi , Kuo Ariel J. , Huang Cho-ying
Published: February 2020

Epiphytic bryophytes (EB) are some of the most commonly found plant species in tropical montane cloud forests, and they play a disproportionate role in influencing the terrestrial hydrological and nutrient cycles. However, it is difficult to estimate the abundance of EB due to the nature of their “epiphytic” habitat. This study proposes an allometric scaling approach to measure EB biomass, implemented in 16,773 ha tropical montane cloud forests of northeastern Taiwan. A general allometry was developed to estimate EB biomass of 100 cm2circular-shaped mats (n = 131) and their central depths. A point-intercept instrument was invented to measure the depths of EB along tree trunks (n = 210) below 3-m from the ground level (sampled stem surface area [SSA]) in twenty-one 30 × 30 m plots. Biomass of EB of each point measure was derived using the general allometry and was aggregated across each SSA, and its performance was evaluated. Total EB biomass of a tree was estimated by referring to anin-situconversion model and was interpolated for all trees in the plots (n = 1451). Finally, we assessed EB biomass density at the plot scale and preliminarily estimated EB biomass of the study region. The general EB biomass-depth allometry showed that the depth of an EB mat was a salient variable for biomass estimation (R2= 0.72,p< 0.001). The performance of upscaling from mats to SSA was satisfactory, which allowed us to further estimate mean (± standard deviation) EB biomass of the 21 plots (272 ± 104 kg ha-1) and to provide preliminary estimation of the total EB biomass of 4562 Mg for the study region. Since a significant relationship between tree size and EB abundance is commonly found, regional EB biomass may be mapped by integrating our method and three-dimensional airborne data.
Epigenetic mediation of the onset of reproduction in a songbird
Authors: Lindner Melanie , Laine Veronika N. , Verhagen Irene , Viitaniemi Heidi M. , Visser Marcel E. , van Oers Kees , Husby Arild
Published: February 2020

Climate change significantly impacts natural populations, particularly phenology traits, like the seasonal onset of reproduction in birds. This impact is mainly via plastic responses in phenology traits to changes in the environment, but the molecular mechanism mediating this plasticity remains elusive. Epigenetic modifications can mediate plasticity and consequently constitute promising candidates for mediating phenology traits. Here, we used genome-wide DNA methylation profiles of individual great tit (Parus major) females that we blood sampled repeatedly throughout the breeding season. We demonstrate rapid and directional variation in DNA methylation within the regulatory region of genes known to play key roles in avian reproduction that are in line with observed changes in gene expression in chickens. Our findings provide an important step towards unraveling the molecular mechanism mediating a key life history trait, an essential knowledge-gap for understanding how natural populations may cope with future climate change.

IMPACT SUMMARY

Natural populations are increasingly challenged by changing environmental conditions like global increases in temperature. A key way for species to adapt to global warming is via phenotypic plasticity, i.e. the ability to adjust the expression of traits to the environment. We, however, know little about how the environment can interact with an organism’s genetic make-up to shape its trait value. Epigenetic marks are known to vary with the environment and can modulate the expression of traits without any change in the genetic make-up and therefore have the potential to mediate phenotypic plasticity.

To study the role of epigenetics for phenotypic plasticity, we here focus on the great tit (Parus major), a species that is strongly affected by global warming and plastic for temperature in an essential phenology trait, the seasonal onset of egg laying. As a first step, we investigated whether great tit females show within-individual and short-term variation in DNA methylation that corresponds to changes in the reproductive state of females. We therefore housed breeding pairs in climate-controlled aviaries to blood sample each female repeatedly throughout the breeding season and used these repeated samples for methylation profiling.

We found rapid and directional variation in DNA methylation at the time females prepared to initiate egg laying that is located within the regulatory region of genes that have previously described functions for avian reproduction. Although future work is needed to establish a causal link between the observed temporal variation in DNA methylation and the onset of reproduction in female great tits, our work highlights the potential role for epigenetic modifications in mediating an essential phenology trait that is sensitive to temperatures.