bluuebunny/crossref_metadata_2025 huggingface dataset:
Reproducible Discovery of Cell-Binding Peptides “Lost” in Bulk Amplification via Emulsion Amplification in Phage Display Panning
Authors: Matochko Wadim L. , Deiss Frédérique , Yang Yang , Derda Ratmir
Published: November 2021

Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naïve phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional ‘bulk’ amplification.

Abstract Figure

Find Ligands in Droplets

Canonical phage display selection of ligands for breast cancer cells, which uses bulk amplification (BA) of phage library, reproducibly identified peptide ligands from a ~0.0001% sub-population of the library, which harbors fast-growing phage. Replacing BA by emulsion-amplification (EmA) altered the selection landscape and yielded cell-binding ligands not accessible to conventional phage-display select
The Temporal Pattern of Synaptic Activation Determines the Longevity of Structural Plasticity at Single Dendritic Spines
Authors: Argunsah Ali Özgür , Israely Inbal
Published: November 2021

Individual synapses are the points at which information is passed between neurons, yet it is unknown how the diverse patterns of activity that are observed in vivo effect plasticity at the level of single inputs. Here, we aimed to determine what are the structural plasticity consequences of naturalistic patterns of activity at single spines, as these reflect changes in synaptic efficacy. Utilizing two- photon fluorescence imaging and glutamate uncaging, we studied structural plasticity of individual CA1 hippocampal dendritic spines using activation patterns sampled from a Poisson distribution, which resemble endogenous firing patterns from their CA3 inputs. We found that while the majority of inputs initially undergo structural changes, the robustness of this plasticity is determined by the timing structure of the Poisson sampled naturalistic stimulation patterns. Further, we found that structural plasticity elicited by these naturalistic patterns is both NMDAR and protein synthesis dependent, consistent with requirements for other forms of plasticity. Lastly, we found that during the delivery of naturalistic activity patterns, spines underwent rapid and dynamic structural growth that predicted the longevity of plasticity, which was not the case during non-naturalistic stimulation protocols. These data suggest that dendritic spines are able to integrate incoming temporal information and accordingly modulate the longevity of plasticity that is induced.

Highlights

Naturalistic stimulation of single dendritic spines of CA1 hippocampal neurons induces long lasting structural plasticity that depends on the temporal distribution of the synaptic events.

Structural plasticity induced by naturalistic stimulation patterns requires NMDA receptor activation and new protein-synthesis.

Rapid spine structural dynamics during naturalistic activity, but not regular patterns, predict the longevity of subsequent structural plasticity.
A prior exposure to Serratia marcescens or xenobiotics primes Drosophila enterocytes against a recurring cytoplasmic purge
Authors: Terreri Simone , Sina-Rahme Bechara , Pais Inês , Socha Catherine , Lestradet Matthieu , Yamba Miriam , Schirmeier Stefanie , Lee Kwang-Zin , Ferrandon Dominique
Published: November 2021

The cytoplasmic extrusion of enterocytes is a fast response to an exposure to pore-forming toxin (PFT)-producing bacteria whereby their apical cytoplasm is extruded into the intestinal lumen. As a result of this purge, the intestinal epithelium becomes thin prior to a subsequent recovery. We report here that the ingestion of ethanol or caffeine induces a similar response, which suggests that a common purging process is triggered by bacterial toxins and abiotic toxicants. We also delineate an additional mechanism that is initiated by these stimuli that we refer to as priming. The initial exposure of the intestinal epithelium to either PFT or xenobiotics protects enterocytes against a further round of purging upon a second bacterial infection. Priming prevents the epithelium from being persistently thin in the context of chronic intestinal infections. We have identified the upper part of the p38b MAPK pathway as well as the homeobox-containing transcription factors E5/EMS as being required for priming and not for the regrowth of enterocytes after the cytoplasmic purge. Unexpectedly, the priming process appears to function cell-nonautonomously. Our findings suggest that the cytoplasmic purge extrusion has been selected because it constitutes a fast reaction to accidental exposure to bacterial toxins or toxicants.
ClinGen CDH1 specifications for the ACMG/AMP guidelines: improvement of germline variant clinical assertions and updated curation guidelines
Authors: Luo Xi , Maciaszek Jamie L. , Thompson Bryony A. , Leong Huei San , Dixon Katherine , Sousa Sónia , Anderson Michael , Roberts Maegan E. , Lee Kristy , Spurdle Amanda B. , Mensenkamp Arjen R. , Brannan Terra , Pardo Carolina , Zhang Liying , Pesaran Tina , Wei Sainan , Fasaye Grace-Ann , Kesserwan Chimene , Shirts Brian H. , Davis Jeremy L. , Oliveira Carla , Plon Sharon E. , Schrader Kasmintan A. , Karam Rachid
Published: November 2021

Purpose

The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations for effective medical care. In addition, the CDH1 VCEP continues to update these specifications in keeping with evolving clinical practice and variant interpretation guidelines.

Methods

CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen, and expert knowledge from ongoing CDH1 variant curations. Trained biocurators curated 273 variants using updated CDH1 interpretation guidelines and incorporated published and unpublished data provided by diagnostic laboratories. All variants were reviewed by the ClinGen VCEP and classifications submitted to ClinVar.

Results

Updated CDH1-specific variant interpretation guidelines include eleven major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36/37) of variants with ClinVar conflicting interpretations were resolved into benign, likely benign, likely pathogenic, or pathogenic, and 35% (15/43) of VUS were resolved into benign or likely benign. Overall, 88% (239/273) of curated variants had non-VUS classifications.

Conclusion

The development and evolution of CDH1-specific criteria by the expert panel results in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene.
Transcriptional integration of distinct microbial and nutritional signals by the small intestinal epithelium
Authors: Lickwar Colin R. , Davison James M. , Kelly Cecelia , Mercado Gilberto Padilla , Wen Jia , Davis Briana R. , Tillman Matthew C. , Semova Ivana , Andres Sarah F. , Vale Goncalo , McDonald Jeffrey G. , Rawls John F.
Published: November 2021

To preserve its physiologic functions, the intestine must interpret and adapt to complex combinations of stimuli from dietary and microbial sources. However, the transcriptional strategies by which the intestinal epithelium integrates and adapts to dietary and microbial information remains unresolved. We compared adult mice reared germ free (GF) or conventionalized with a microbiota (CV) either fed normally or after a single high-fat meal (HFM). Jejunal epithelium preparations were queried using genomewide assays for RNA-seq, the activating histone mark H3K27ac ChIP-seq, and ChIP-seq of the microbially-responsive transcription factor HNF4A. We identified distinct nutritional and microbial responses at certain genes, but also apparent simultaneous influence of both stimuli at many other loci and regulatory regions. Increased expression levels and H3K27ac enrichment following HFM at a subset of these sites was dependent on microbial status. H3K27ac sites that were preferentially increased by HFM in the presence of microbes neighbor lipid anabolism and proliferation genes as well as intestinal stem cell (ISC) markers, were usually active only in ISCs, and were not HNF4A targets. In contrast, H3K27ac sites that were preferentially increased by HFM in the absence of microbes neighbored targets of the nuclear receptor and energy homeostasis regulator PPARA, were frequently accessible only in enterocytes, and were HNF4A bound. These results reveal that HNF4A supports a differentiated enterocyte and FAO program in GF, and that suppression of HNF4A by the combination of microbes and HFM may result in preferential activation of IEC proliferation programs. Microbial and nutritional responses are therefore integrated with some of the same transcriptional programs that regulate intestinal proliferation and differentiation.
Dorsal striatum coding for the timely execution of action sequences
Authors: Martinez María Cecilia , Zold Camila Lidia , Coletti Marcos Antonio , Murer Mario Gustavo , Belluscio Mariano Andrés
Published: November 2021

The automatic initiation of actions can be highly functional. But occasionally these actions cannot be withheld and are released at inappropriate times, impulsively. Striatal activity has been shown to participate in the timing of action sequence initiation and it has been linked to impulsivity. Using a self-initiated task, we trained adult male rats to withhold a rewarded action sequence until a waiting time interval has elapsed. By analyzing neuronal activity we show that the striatal response preceding the initiation of the learned sequence is strongly modulated by the time subjects wait before eliciting the sequence. Interestingly, the modulation is steeper in adolescent rats, which show a strong prevalence of impulsive responses compared to adults. We hypothesize this anticipatory striatal activity reflects the animals’ subjective reward expectation, based on the elapsed waiting time, while the steeper waiting modulation in adolescence reflects age-related differences in temporal discounting, internal urgency states or explore-exploit balance.
Oxytocin promotes epicardial cell activation and heart regeneration after cardiac injury
Authors: Wasserman Aaron H. , Huang Amanda R. , Lewis-Israeli Yonatan R. , Dooley McKenna D. , Mitchell Allison L. , Venkatesan Manigandan , Aguirre Aitor
Published: November 2021

Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide, and frequently leads to massive heart injury and the loss of billions of cardiac muscle cells and associated vasculature. Critical work in the last two decades demonstrated that these lost cells can be partially regenerated by the epicardium, the outermost mesothelial layer of the heart, in a process that highly recapitulates its role in heart development. Upon cardiac injury, mature epicardial cells activate and undergo an epithelial-mesenchymal transition (EMT) to form epicardial-derived progenitor cells (EpiPCs), multipotent progenitors that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. In mammals, this process alone is insufficient for significant regeneration, but it might be possible to prime it by administering specific reprogramming factors, leading to enhanced EpiPC function. Here, we show that oxytocin (OXT), a hypothalamic neuroendocrine peptide, induces epicardial cell proliferation, EMT, and migration in a model of human induced pluripotent stem cell (hiPSC)-derived epicardial cells. In addition, we demonstrate that OXT is produced after cardiac cryoinjury in zebrafish, and that it elicits significant epicardial activation promoting heart regeneration. Oxytocin signaling is also critical for proper epicardium development in zebrafish embryos. The above processes are significantly impaired when OXT signaling is inhibited chemically or genetically through RNA interference. RNA sequencing data suggests that the transforming growth factor beta (TGF-β) pathway is the primary mediator of OXT-induced epicardial activation. Our research reveals for the first time an evolutionary conserved brain-controlled mechanism inducing cellular reprogramming and regeneration of the injured mammalian and zebrafish heart, a finding that could contribute to translational advances for the treatment of cardiac injuries.
Concerted cellular responses to type I interferon propel memory impairment associated with amyloid β plaques
Authors: Roy Ethan R. , Chiu Gabriel , Li Sanming , Propson Nicholas E. , Zheng Hui , Cao Wei
Published: November 2021

Despite well-documented maladaptive neuroinflammation in Alzheimer’s disease (AD), the principal signal that drives memory and cognitive impairment remains elusive. Here, we reveal robust, age-dependent cellular reactions to type I interferon (IFN), an innate immune cytokine aberrantly elicited by β amyloid plaques, and examine their role in cognition and neuropathology relevant to AD in a murine amyloidosis model. Long-term blockade of IFN receptor rescued both memory and synaptic deficits, and also resulted in reduced microgliosis, inflammation, and neuritic pathology. Interestingly, microglia-specific IFN receptor ablation attenuated the loss of post-synaptic terminals, whereas IFN signaling in neural cells contributed to pre-synaptic alteration and plaque accumulation. Intriguingly, IFN pathway activation displayed a strong inverse correlation with cognitive performance, promoting selective synapse engulfment by microglia rather than amyloid plaques. Overall, IFN signaling represents a critical module within the neuroinflammatory network of AD and prompts a concerted cellular state that is detrimental to memory and cognition.
A low-resource reliable pipeline to democratize multi-modal connectome estimation and analysis
Authors: Chung Jaewon , Lawrence Ross , Loftus Alex , Pisner Derek , Kiar Gregory , Bridgeford Eric W. , Roncal William Gray ,   Consortium for Reliability and Reproducibility (CoRR), Chandrashekhar Vikram , Mhembere Disa , Ryman Sephira , Zuo Xi-Nian , Margulies Daniel S. , Craddock R. Cameron , Priebe Carey E. , Jung Rex , Calhoun Vince D. , Caffo Brian , Burns Randal , Milham Michael P. , Vogelstein Joshua T.
Published: November 2021

Connectomics—the study of brain networks—provides a unique and valuable opportunity to study the brain. Research in human connectomics, leveraging functional and diffusion Magnetic Resonance Imaging (MRI), is a resource-intensive practice. Typical analysis routines require significant computational capabilities and subject matter expertise. Establishing a pipeline that is low-resource, easy to use, and off-the-shelf (can be applied across multifarious datasets without parameter tuning to reliably estimate plausible connectomes), would significantly lower the barrier to entry into connectomics, thereby democratizing the field by empowering a more diverse and inclusive community of connectomists. We therefore introduce ‘MRI to Graphs’ (m2g). To illustrate its properties, we usedm2gto process MRI data from 35 different studies (≈ 6,000 scans) from 15 sites without any manual intervention or parameter tuning. Every single scan yielded an estimated connectome that adhered to established properties, such as stronger ipsilateral than contralateral connections in structural connectomes, and stronger homotopic than heterotopic correlations in functional connectomes. Moreover, the connectomes estimated bym2gare more similar within individuals than between them, suggesting thatm2gpreserves biological variability.m2gis portable, and can run on a single CPU with 16 GB of RAM in less than a couple hours, or be deployed on the cloud using its docker container. All code is available onhttps://github.com/neurodata/m2gand documentation is available on docs.neurodata.io/m2g.
Antiviral activity of pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus
Authors: Pedler Rebecca L. , Harris James O. , Speck Peter G.
Published: November 2021

Coronaviruses can cause severe respiratory infections in humans. In this study we assessed the antiviral activity of Pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus, HCoV-229E. An eight-fold reduction in infectivity of HCoV-229E on Huh-7 cells was observed in the presence of 10% C. gigas hemolymph. Antiviral activity of C. gigas hemolymph positively correlated with its concentration and appears to be active during an intracellular stage of HCoV-229E infection.
Isomeric lipid signatures reveal compartmentalised fatty acid metabolism in cancer
Authors: Young Reuben S. E. , Bowman Andrew P. , Tousignant Kaylyn D. , Poad Berwyck L. J. , Gunter Jennifer H. , Philp Lisa K. , Nelson Colleen C. , Ellis Shane R. , Heeren Ron M. A. , Sadowski Martin C. , Blanksby Stephen J.
Published: November 2021

1.0

Cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular fatty acids (FA) and de novo synthesis. Given that oxidation of de novo synthesised FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates - however hitherto FAs were considered part of a common pool. To probe FA metabolic partitioning, cancer cells were supplemented with stable-isotope labelled FAs. Structural analysis of the resulting glycerophospholipids revealed that labelled FAs from uptake were largely incorporated to canonical (sn-)positions on the glycerol backbone. Surprisingly, labelled FA uptake disrupted canonical isomer patterns of the unlabelled lipidome and induced repartitioning of n-3 and n-6 polyunsaturated-FAs into glycerophospholipid classes. These structural changes evidence differences in the metabolic fate of FAs derived from uptake or de novo sources and demonstrate unique signalling and remodelling behaviours usually hidden to conventional lipidomics.

Highlights

Lipid isomers reveal discrete metabolic compartmentalisation in cancer

FAs derived from uptake and de novo synthesis have different metabolic fates

Stearate uptake signals for PUFA (n-3 and n-6) repartitioning between lipid classes

sn-positional isomers are a marker for aberrant lipid metabolism
Long-term forest monitoring unravels constant mortality rise in European forests
Authors: George Jan-Peter , Sanders Tanja GM , Neumann Mathias , Cammalleri Carmelo , Vogt Jürgen V. , Lang Mait
Published: November 2021

⍰

European forests are an important source for timber production, human welfare, income, protection and biodiversity. During the last two decades, Europe has experienced a number of droughts which were exceptionally within the last 500 years both in terms of duration and intensity and these droughts seem to left remarkable imprints in the mortality dynamics of European forests. However, systematic observations on tree decline with emphasis on single species together with high-resolution drought data has been scarce so far so that deeper insights into mortality dynamics and drought occurrence is still limiting our understanding at continental scale.

⍰

Here we make use of the ICP Forest crown defoliation dataset, permitting us to retrospectively monitor tree mortality for four major conifers, two major broadleaves as well as a pooled dataset of nearly all minor tree species in Europe. In total, we analysed more than 3 million observations gathered during the last 25 years and employed a high-resolution drought index which is able to assess soil moisture anomaly based on a hydrological water-balance and runoff model every ten days globally. ⍰We found significant overall and species-specific increasing trends in mortality rates accompanied by decreasing soil moisture. A generalized linear model identified previous-year soil moisture anomaly as the most important driver of mortality patterns in European forests. Significant interactions appeared between previous-year soil moisture and stand water regime in conifers, strongly suggesting that conifers growing at productive sites are more vulnerable under drought.

⍰

We conclude that mortality patterns in European forests are currently reaching a concerning upward trend which could be further accelerated by global change-type droughts.

Key message

Forest mortality has significantly increased over the last 25 years and remained above the long-term mean since 2012.
Multiplexed Single-Molecule Epigenetic Analysis of Plasma-Isolated Nucleosomes for Cancer Diagnostics
Authors: Fedyuk Vadim , Erez Nir , Furth Noa , Beresh Olga , Andreishcheva Ekaterina , Shinde Abhijeet , Jones Daniel , Zakai Barak Bar , Mavor Yael , Peretz Tamar , Hubert Ayala , Cohen Jonathan E , Salah Azzam , Temper Mark , Grinshpun Albert , Maoz Myriam , Zick Aviad , Ron Guy , Shema Efrat
Published: November 2021

The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine, providing information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed EPINUC, a single-molecule multi-parametric assay to comprehensively profile the Epigenetics of Plasma Isolated Nucleosomes, DNA methylation and cancer-specific protein biomarkers. Our system allows high-resolution detection of six active and repressive histone modifications, their ratios and combinatorial patterns, on millions of individual nucleosomes by single-molecule imaging. In addition, it provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins such as mutant p53. Applying this analysis to a cohort of plasma samples detected colorectal cancer at high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue-of-origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multi-layered clinical-relevant information from limited liquid biopsy material, establishing a novel approach for cancer diagnostics.
Substitution load revisited: a high proportion of deaths can be selective
Authors: Matheson Joseph , Exposito-Alonso Moises , Masel Joanna
Published: November 2021

Haldane’s Dilemma refers to the concern that the need for many “selective deaths” to complete a substitution (i.e. selective sweep) creates a speed limit to adaptation. However, discussion of this concern has been marked by confusion, especially with respect to the term “substitution load”. Here we distinguish different historical lines of reasoning, and identify one, focused on finite reproductive excess and the proportion of deaths that are “selective” (i.e. causally contribute to adaptive allele frequency changes), that has not yet been fully addressed. We develop this into a more general theoretical model that can apply to populations with any life history, even those for which a generation or even an individual are not well defined. The actual speed of adaptive evolution is coupled to the proportion of deaths that are selective. The degree to which reproductive excess enables a high proportion of selective deaths depends on the details of when selection takes place relative to density regulation, and there is therefore no general expression for a speed limit. To make these concepts concrete, we estimate both reproductive excess, and the proportion of deaths that are selective, from a dataset measuring survival of 517 different genotypes ofArabidopsis thalianagrown in eight different environmental conditions. In this dataset, a much higher proportion of deaths contribute to adaptation, in all environmental conditions, than the 10% cap that was anticipated as substantially restricting adaptation during historical discussions of speed limits.

LAY SUMMARY

The influential neutral theory of molecular evolution was predicated on theoretical arguments that adaptation is subject to a speed limit. We resolve confusions regarding historical speed limit arguments, which depend on differences in fitness, not variance (differences in fitness squared). We generalize the underlying concepts of selective deaths and reproductive excess to populations with any life cycle, even those for which an “individual” and hence generation and fitness, are poorly defined. We apply the revised theory to Arabidopsis data, demonstrating the potential for future related experiments.
Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation
Authors: Takele Yegnasew , Adem Emebet , Franssen Susanne Ursula , Womersley Rebecca , Kaforou Myrsini , Levin Michael , Müller Ingrid , Cotton James Anthony , Kropf Pascale
Published: November 2021

Visceral leishmaniasis (VL) is a neglected tropical disease that causes substantial morbidity and mortality and is a growing health problem in Ethiopia, where this study took place. Most individuals infected with Leishmania donovani parasites will stay asymptomatic, but some develop VL that, if left untreated, is almost always fatal. This stage of the disease is associated with a profound immunosuppression, characterised by impaired production of Interferonγ (IFNγ), a cytokine that plays a key role in the control of Leishmania parasites, and high expression levels of an inhibitory receptor, programmed cell death 1 (PD1) on CD4+ T cells. Here, we tested the contribution of the interaction between the immune checkpoint PD1 and its ligand PDL-1 on the impaired production of IFNγ in VL patients. Our results show that in the blood of VL patients, not only CD4+, but also CD8+ T cells express high levels of PD1 at the time of VL diagnosis. Next, we identified PDL-1 expression on different monocyte subsets and neutrophils and show that PDL-1 levels were significantly increased in VL patients. PD1/PDL-1 inhibition resulted in significantly increased production of IFNγ, suggesting that therapy using immune checkpoint inhibitors might improve disease control in these patients.
Penalized generalized estimating equations for relative risk regression with applications to brain lesion data
Authors: Kindalova Petya , Veldsman Michele , Nichols Thomas E. , Kosmidis Ioannis
Published: November 2021

Motivated by a brain lesion application, we introduce penalized generalized estimating equations for relative risk regression for modelling correlated binary data. Brain lesions can have varying incidence across the brain and result in both rare and high incidence outcomes. As a result, odds ratios estimated from generalized estimating equations with logistic regression structures are not necessarily directly interpretable as relative risks. On the other hand, use of log-link regression structures with the binomial variance function may lead to estimation instabilities when event probabilities are close to 1. To circumvent such issues, we use generalized estimating equations with log-link regression structures with identity variance function and unknown dispersion parameter. Even in this setting, parameter estimates can be infinite, which we address by penalizing the generalized estimating functions with the gradient of the Jeffreys prior.

Our findings from extensive simulation studies show significant improvement over the standard log-link generalized estimating equations by providing finite estimates and achieving convergence when boundary estimates occur. The real data application on UK Biobank brain lesion maps further reveals the instabilities of the standard log-link generalized estimating equations for a large-scale data set and demonstrates the clear interpretation of relative risk in clinical applications.
Tau Differentially Regulates the Transport of Early Endosomes and Lysosomes
Authors: Balabanian Linda , Lessard Dominique V. , Yaninska Pamela , Stevens Piper W. , Wiseman Paul W. , Berger Christopher L. , Hendricks Adam G.
Published: November 2021

Microtubule-associated proteins (MAPs) modulate the motility of kinesin and dynein along microtubules to control the transport of vesicles and organelles. The neuronal MAP tau inhibits kinesin-dependent transport. Phosphorylation of tau at tyrosine 18 by fyn kinase results in weakened inhibition of kinesin-1. We examined the motility of early endosomes and lysosomes in cells expressing wild-type (WT) tau and phosphomimetic Y18E tau. Lysosome motility is strongly inhibited by tau. Y18E tau preferentially inhibits lysosomes in the cell periphery, while centrally located lysosomes are less affected. Early endosomes are more sensitive to tau than lysosomes, and are inhibited by both WT and Y18E tau. Our results show that different cargoes have disparate responses to tau, likely governed by the types of kinesin motors driving their transport. In support of this model, kinesin-1 and -3 are strongly inhibited by tau while kinesin-2 and dynein are less affected. In contrast to kinesin-1, we find that kinesin-3 is strongly inhibited by phosphorylated tau.
A system for multiplexed selection of aptamers with exquisite specificity without counter-selection
Authors: Yoshikawa Alex M. , Wan Leighton , Zheng Liwei , Eisenstein Michael , Soh H. Tom
Published: November 2021

Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counter-selection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counter-selection parameters. As a result, counter-selection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel, while also eliminating the need for counter-selection. We employ a platform based on a modified benchtop sequencer to conduct a massively-parallel aptamer screening process that enables the selection of highly-specific aptamers against multiple structurally similar molecules in a single experiment, without any counter-selection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes, and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.

Significance statement

Aptamers offer the exciting potential to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has been proven challenging using the conventional process of counter-selection. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers towards a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counter-selection. As exemplars, we generated aptamers with high affinity and specificity towards three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.
Feline Papillomavirus Isolate P20 Assembled from Metagenomic Data Isolated from the Human Skin of a House Cat Owner
Authors: Graham Ema Helene , Adamowicz Michael S. , Angeletti Peter , Clarke Jennifer , Fernando Samodha , Herr Joshua R
Published: November 2021

A feline papillomavirus was assembled from metagenomic sequencing data collected from the human skin of a house cat owner. This circular papillomavirus strain P20 is 8069 bp in length, has a GC content of 54.38%, and displays genome organization typical of feline papillomaviruses with six annotated protein coding regions. The genome exhibits approximately 75% sequence similarity to other feline papillomavirus genomes.
Impact of antibiotics to off-target infant gut microbiota and resistance genes in cohort studies
Authors: Lebeaux Rebecca M. , Madan Juliette C. , Nguyen Quang P. , Coker Modupe O. , Dade Erika F. , Moroishi Yuka , Palys Thomas J. , Ross Benjamin D. , Pettigrew Melinda M. , Morrison Hilary G. , Karagas Margaret R. , Hoen Anne G.
Published: November 2021

Background

Young children are frequently exposed to antibiotics for otitis media and respiratory infections, with the potential for collateral consequences on the gut microbiome. The impact of antibiotic exposures to off-target microbes (i.e., bacteria not targeted by antibiotic treatment) and antibiotic resistance genes (ARGs) is unknown.

Methods

We used metagenomic sequencing data from paired stool samples collected prior to antibiotic exposure and at 1 year from over 200 infants and a difference-in-differences approach to assess the relationship between subsequent exposures and the abundance or compositional diversity of off-target microbes and ARGs while adjusting for covariates.

Results

By 1 year, the relative abundance of multiple species and ARGs differed by antibiotic exposure. Compared to infants never exposed to antibiotics, Bacteroides vulgatus relative abundance increased by 1.72% (95%CI:0.19,3.24) while Bacteroides fragilis decreased by 1.56% (95%CI:-4.32,1.21). Bifidobacterium species also exhibited opposing trends suggesting differential antibiotic selection. Overall, antibiotic exposure was associated with a dose-dependent decrease in alpha diversity of off-target microbes. ARGs associated with antibiotic exposure included class A beta-lactamase gene CfxA6. Among infants attending day care, Escherichia coli and ARG abundance were both positively associated with antibiotic use.

Conclusion

Further quantifying impacts to off-target microbes and ARGs has implications for antibiotic stewardship

Impact

Infants are frequently exposed to antibiotics for respiratory illnesses, but the extent of impact to off-target gut microbiota and antibiotic resistance genes is unknown.

We quantified these impacts in 2 cohort studies using a difference-in-differences approach. Novel to the microbiome space, this enabled us to use pre/post antibiotic data to emulate a randomized controlled trial.

Compared to infants unexposed to antibiotics between six weeks and 1 year, the relative abundance of multiple off-target species and antibiotic resistance genes was altered.

This research highlights the need to consider the microbiome in antibiotic stewardship and offers a new framework for quantifying change.
Marker-free coselection for successive rounds of prime editing in human cells
Authors: Levesque Sébastien , Mayorga Diana , Fiset Jean-Philippe , Goupil Claudia , Duringer Alexis , Loiselle Andréanne , Bouchard Eva , Agudelo Daniel , Doyon Yannick
Published: November 2021

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we designed a robust coselection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ATPase). We readily engineered highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process revealed that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific coselection strategies.
Loop-extrusion and polymer phase-separation can co-exist at the single-molecule level to shape chromatin folding
Authors: Conte Mattia , Irani Ehsan , Chiariello Andrea M. , Abraham Alex , Bianco Simona , Esposito Andrea , Nicodemi Mario
Published: November 2021

Loop-extrusion and phase-separation have been proposed as mechanisms that shape chromosome large-scale spatial organization. It is unclear, however, how they perform relative to each other in explaining chromatin architecture data and whether they compete or co-exist at the single-molecule level. Here, we compare models of polymer physics based on loop-extrusion and phase-separation, as well as models where both mechanisms act simultaneously in a single molecule, against multiplexed FISH data available in human loci in IMR90 and HCT116 cells. We find that the different models recapitulate bulk Hi-C and average microscopy data. Single-molecule chromatin conformations are also well captured, especially by phase-separation based models that better reflect the experimentally reported segregation in globules of the considered genomic loci and their cell-to-cell structural variability. Such a variability is consistent with two main concurrent causes: single-cell epigenetic heterogeneity and an intrinsic thermodynamic conformational degeneracy of folding. Overall, the model combining loop-extrusion and polymer phase-separation provides a very good description of the data, particularly higher-order contacts, showing that the two mechanisms can co-exist in shaping chromatin architecture in single cells.
Manipulating neural dynamics to tune motion detection
Authors: Gonzalez-Suarez Aneysis D. , Zavatone-Veth Jacob A. , Chen Juyue , Matulis Catherine A. , Badwan Bara A. , Clark Damon A.
Published: November 2021

Neurons integrate excitatory and inhibitory signals to produce their outputs, but the role of input timing in this integration remains poorly understood. Motion detection is a paradigmatic example of this integration, since theories of motion detection rely on different delays in visual signals. These delays allow circuits to compare scenes at different times to calculate the direction and speed of motion. It remains untested how response dynamics of individual cell types drive motion detection and velocity sensitivity. Here, we sped up or slowed down specific neuron types in Drosophila’s motion detection circuit by manipulating ion channel expression. Altering the dynamics of individual neurons upstream of motion detectors changed their integrating properties and increased their sensitivity to fast or slow visual motion, exposing distinct roles for dynamics in tuning directional signals. A circuit model constrained by data and anatomy reproduced the observed tuning changes. Together, these results reveal how excitatory and inhibitory dynamics jointly tune a canonical circuit computation.
Mathematical modeling of the molecular switch of TNFR1-mediated signaling pathways using Petri nets
Authors: Amstein Leonie K. , Ackermann Jörg , Hannig Jennifer , Ðikić Ivan , Fulda Simone , Koch Ina
Published: November 2021

The paper describes a mathematical model of the molecular switch of cell survival, apoptosis, and necroptosis in cellular signaling pathways initiated by tumor necrosis factor 1. Based on experimental findings in the current literature, we constructed a Petri net model in terms of detailed molecular reactions for the molecular players, protein complexes, post-translational modifications, and cross talk. The model comprises 118 biochemical entities, 130 reactions, and 299 connecting edges. Applying Petri net analysis techniques, we found 279 pathways describing complete signal flows from receptor activation to cellular response, representing the combinatorial diversity of functional pathways.120 pathways steered the cell to survival, whereas 58 and 35 pathways led to apoptosis and necroptosis, respectively. For 65 pathways, the triggered response was not deterministic, leading to multiple possible outcomes. Based on the Petri net, we investigated the detailed in silico knockout behavior and identified important checkpoints of the TNFR1 signaling pathway in terms of ubiquitination within complex I and the gene expression dependent on NF-κB, which controls the caspase activity in complex II and apoptosis induction.
Altered expression of Api5 affects breast carcinogenesis by modulating FGF2 signalling
Authors: Abhijith K , Panda Debiprasad , Malaviya Radhika , Gaidhani Gautami , Lahiri Mayurika
Published: November 2021

Apoptosis or programmed cell death plays a vital role in maintaining homeostasis and, therefore, is a tightly regulated process. Deregulation of apoptosis signalling can favour carcinogenesis. Apoptosis inhibitor 5 (Api5), an inhibitor of apoptosis, is upregulated in cancers. Interestingly, Api5 is shown to regulate both apoptosis and cell proliferation. To address the precise functional significance of Api5 in carcinogenesis here we investigate the role of Api5 in breast carcinogenesis.

Consistently, in-silico analysis revealed elevated levels of Api5 transcript in breast cancer patients which correlated with poor prognosis. Overexpression of Api5 in non-tumorigenic breast acinar cultures resulted in increased proliferation and cells exhibited a partial EMT-like phenotype with higher migratory potential and disruption in cell polarity. Furthermore, during acini development, the influence of Api5 is mediated via the combined action of FGF2 activated PDK1-Akt/cMYC signalling and Ras-ERK pathways. Conversely, Api5 knock-down downregulated FGF2 signalling leading to reduced proliferation and diminished in vivo tumorigenic potential of the breast cancer cells. Thus, taken together, our study identifies Api5 as a central player involved in regulating multiple events during breast carcinogenesis.
Epithelial-mesenchymal plasticity induced by discontinuous exposure to TGFβ1 promotes tumour growth
Authors: Santos Mafalda , Ferreira Marta , Oliveira Patrícia , Mendes Nuno , André Ana , Vieira André F. , Nunes Joana B. , Carvalho Joana , Rocha Sara , Azevedo Mafalda , Ferreira Daniel , Reis Inês , Vinagre João , Paredes Joana , Heravi-Moussavi Alireza , Lima Jorge , Máximo Valdemar , Burleigh Angela , Roskelley Calvin , Fátima Carneiro Maria de , Huntsman David , Oliveira Carla
Published: November 2021

Transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression. We hypothesize that EMT followed by MET promotes cell population heterogeneity favouring tumour growth. We developed an EMT model by on/off exposure of epithelial EpH4 cells (E-cells) to TGFβ1 that mimics phenotypic EMT (M-cells) and MET. We aimed at understanding whether phenotypic MET is accompanied by molecular and functional reversion back to epithelia, by using RNA sequencing, Immunofluorescence (IF), proliferation, wound healing, focus formation and mamosphere formation assays, as well as cell-xenografts in nude mice. Phenotypic reverted-epithelial cells (RE-cells), obtained after MET induction, presented pure epithelial morphology and proliferation rate resembling E-cells. However, RE transcriptomic profile and IF staining of epithelial and mesenchymal markers revealed a unique and heterogeneous mixture of cell-subpopulations, with high self-renewal ability fed by oxidative phosporylation. RE-cells heterogeneity is stably maintained for long periods after TGFβ1 removal, both in vitro and in large derived tumours in nude mice. Overall, we show that phenotypic reverted-epithelial cells (RE-cells) do not return to the molecular and functional epithelial state, present mesenchymal features related with aggressiveness and cellular heterogeneity that favour tumour growth in vivo. This work strengthens epithelial cells reprogramming and cellular heterogeneity fostered by inflammatory cues as a tumour-growth promoting factor in vivo.
RBBP6 activates the pre-mRNA 3’-end processing machinery in humans
Authors: Boreikaite Vytaute , Elliott Thomas , Chin Jason , Passmore Lori A
Published: November 2021

3’-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3’-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3’-endonuclease activity of human CPSF with purified proteins. This requires the sevensubunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.
Bright, fluorogenic and photostable avidity probes for RNA imaging
Authors: Bühler Bastian , Benderoth Anja , Englert Daniel , Grün Franziska , Schokolowski Janin , Jäschke Andres , Sunbul Murat
Published: November 2021

Fluorescent light-up aptamers (FLAPs) emerged as valuable tools to visualize RNA, but are mostly limited by poor brightness, low photostability and high fluorescence background. In this study, we combine bivalent (silicon) rhodamine fluorophores with dimeric FLAPs to yield bright and photostable complexes with low picomolar dissociation constants. Our avidity-based approach resulted in extreme binding strength and high fluorogenicity, enabling single mRNA tracking in living cells.
JAK-STAT Signaling Enables Lineage Plasticity-driven AR Targeted Therapy Resistance
Authors: Deng Su , Wang Choushi , Wang Yunguan , Xu Yaru , Li Xiaoling , Johnson Nickolas A , Lo U-Ging , Xu Lingfan , Gonzalez Julisa , Metang Lauren A , Ye Jianfeng , Tirado Carla Rodriguez , Rodarte Kathia , Xie Zhiqun , Arana Carlos , Annamalai Valli , Hsieh Jer-Tsong , Vander Griend Donald J. , Li Bo , Wang Tao , Mu Ping
Published: November 2021

Emerging evidence indicates that various cancers can gain resistance to targeted therapies by acquiring lineage plasticity. Although various genomic and transcriptomic aberrations correlate with lineage plasticity-driven resistance, the molecular mechanisms of acquiring lineage plasticity have not been fully elucidated. Through integrated transcriptomic and single cell RNA-Seq (scRNA-Seq) analysis of more than 80,000 cells, we reveal for the first time that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is a crucial executor in promoting lineage plasticity-driven AR targeted therapy resistance in prostate cancer. Ectopic activation of JAK-STAT signaling is specifically required for the AR targeted therapy resistance of subclones expressing multilineage, stem-like and epithelial-to-mesenchymal transition (EMT) lineage transcriptional programs and represents a potential therapeutic target for overcoming AR targeted therapy resistance.

One-Sentence Summary

JAK-STAT signaling is a crucial executor in promoting lineage plasticity-driven AR therapy resistance in prostate cancer.