bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyper-adaptation in the Human Brain: Functional and structural changes in the foot section of the primary motor cortex in a top wheelchair racing Paralympian
Authors: Morita Tomoyo , Hirose Satoshi , Kimura Nodoka , Takemura Hiromasa , Asada Minoru , Naito Eiichi
Published: March 2022

The human brain has the capacity to drastically alter its somatotopic representations in response to congenital or acquired limb deficiencies and dysfunctions. The main purpose of the present study was to elucidate such extreme adaptability in the brain of an active top wheelchair racing Paralympian (participant P1) who has congenital paraplegia (dysfunction of bilateral lower limbs). Participant P1 has undergone long-term wheelchair racing training using bilateral upper limbs and has won a total of 19 medals in six consecutive summer Paralympic games as of 2021. We examined the functional and structural changes in the foot section of the primary motor cortex (M1) in participant P1 as compared to able-bodied control participants. We also examined the functional and structural changes in three other individuals (participants P2, P3, and P4) with acquired paraplegia, who also had long-term non-use period of the lower limbs and had undergone long-term training for wheelchair sports (but not top athletes at the level of participant P1). We measured brain activity in all the participants using functional magnetic resonance imaging (MRI) when bimanual wrist extension-flexion movement was performed, and the structural MRI images were collected. Compared to 37 control participants, participant P1 showed significantly greater activity in the M1 foot section during the bimanual task, and significant local GM expansion in this section. Significantly greater activity in the M1 foot section was also observed in participant P4, but not in P2 and P3, and the significant local GM expansion was observed in participant P2, but not in P3 and P4. Thus, functional or structural change was observed in an acquired paraplegic participant, but was not observed in all the paraplegic participants. The functional and structural changes typically observed in participant P1 may represent extreme adaptability of the human brain. We discuss the results in terms of a new idea of hyper-adaptation.
Phenotype to genotype mapping using supervised and unsupervised learning
Authors: Pastore Vito Paolo , Oke Ashwini , Capponi Sara , Elnatan Daniel , Fung Jennifer , Bianco Simone
Published: March 2022

The relationship between the genotype, the genetic instructions encoded into a genome, and phenotype, the macroscopic realization of such instructions, remains mostly uncharted. In addition, tools able to uncover the connection between the phenotype with a specific set of responsible genes are still under definition. In this work, we focus on yeast organelles called vacuoles, which are cell membrane compartments that vary size and shape in response to various stimuli, and we develop a framework relating changes of cellular morphology to genetic modification. The method is a combination of convolutional neural network (CNN) and an unsupervised learning pipeline, which employs a deep-learning based segmentation, classification, and anomaly detection algorithm. From the live 3D fluorescence vacuole images, we observe that different genetic mutations generate distinct vacuole phenotypes and that the same mutation might correspond to more than one vacuole morphology. We trained a Unet architecture to segment our cellular images and obtain precise, quantitative information in 2D depth-encoded images. We then used an unsupervised learning approach to cluster the vacuole types and to establish a correlation between genotype and vacuole morphology. Using this procedure, we obtained 4 phenotypic groups. We extracted a set of 131 morphological features from the segmented vacuoles images, reduced to 50 after a tree-based feature selection. We obtained a purity of 85% adopting a Fuzzy K-Means based algorithm on a random subset of 880 images, containing all the detected phenotypic groups. Finally, we trained a CNN on the labels assigned during clustering. The CNN has been used for prediction of a large dataset (6942 images) with high accuracy (80%). Our approach can be applied extensively for live fluorescence image analysis and most importantly can unveil the basic principles relating genotype to vacuole phenotype in yeast cell, which can be thought as a first step for inferring cell designing principles to generate organelles with a specific, desired morphology.
Reference values for amplitude-integrated EEGs in children from 1 month to 17 years of age
Authors: Greve Sandra , Löffelhardt Verena Tamara , Della-Marina Adela , Felderhoff-Müser Ursula , Dohna-Schwake Christian , Bruns Nora
Published: March 2022

Aim

Amplitude-integrated electroencephalography (aEEG) is used to monitor electrocortical activity in critically ill children, but reference values are lacking for patients older than 3.5 months. We aimed to derive reference values for paediatric aEEGs from neurologically healthy children.

Methods

Normal EEGs from awake children aged 1 month to 17 years (213 female, 237 male) without neurological disease or neuroactive medication were retrospectively converted into aEEGs. Two observers manually measured the upper and lower amplitude borders of the C3 – P3, C4 – P4, C3 – C4, P3 – P4, and Fp1 – Fp2 channels of the 10 – 20 system. Percentiles (10th, 25th, 50th, 75th, 90th) were calculated for each age group (< 1 year, 1 year, 2 – 5 years, 6 – 9 years, 10 – 13 years, 14 – 17 years).

Results

Amplitude heights and curves differed between channels without sex-specific differences. During the first 2 years of life, upper and lower amplitudes of all but the Fp1 – Fp2 channel increased and then declined until 17 years. The decline of the upper Fp1 – Fp2 amplitude began at four years, while the lower amplitude declined from the first year of life.

Interpretation

aEEG interpretation must account for age and electrode positions but not for sex in infants and children.

What this paper adds

-

aEEG amplitudes increase in the first years of life and then decline.

-

aEEG amplitudes depend on electrode positions but not on sex.

-

aEEG interpretation must account for age and electrode positions.
Wochenende - modular and flexible alignment-based shotgun metagenome analysis
Authors: Rosenboom Ilona , Scheithauer Tobias , Friedrich Fabian C. , Pörtner Sophia , Hollstein Lisa , Pust Marie-Madlen , Sifakis Konstantinos , Wehrbein Tom , Rosenhahn Bodo , Wiehlmann Lutz , Chhatwal Patrick , Tümmler Burkhard , Davenport Colin F.
Published: March 2022

Background

Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively.

Results

Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization, as well as integration and subsequent plotting of pipeline results. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome.

Conclusion

Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende.
The genetic and biochemical determinants of mRNA degradation rates in mammals
Authors: Agarwal Vikram , Kelley David
Published: March 2022

Background

Degradation rate is a fundamental aspect of mRNA metabolism, and the factors governing it remain poorly characterized. Understanding the genetic and biochemical determinants of mRNA half-life would enable a more precise identification of variants that perturb gene expression through post-transcriptional gene regulatory mechanisms.

Results

Here, we establish a compendium of 54 human and 27 mouse transcriptome-wide mRNA decay rate datasets. A meta-analysis of these data identified a prevalence of technical noise and measurement bias, induced partially by the underlying experimental strategy. Correcting for these biases allowed us to derive more precise, consensus measurements of half-life which exhibit enhanced consistency between species. We trained substantially improved statistical models based upon genetic and biochemical features to better predict half-life and characterize the factors molding it. Our state-of-the-art model, Saluki, is a hybrid convolutional and recurrent deep neural network which relies only upon an mRNA sequence annotated with coding frame and splice sites to predict half-life (r=0.77). Saluki predicts the impact of RNA sequences and genetic mutations therein on mRNA stability, in agreement with functional measurements derived from massively parallel reporter assays.

Conclusions

Our work produces a more robust “ground truth” with regards to transcriptome-wide mRNA half-lives in mammalian cells. Using these consolidated measurements, we trained a model that is over 50% more accurate in predicting half-life from sequence than existing models. Our best model, Saluki, succinctly captures many of the known determinants of mRNA half-life and can be rapidly deployed to predict the functional consequences of arbitrary mutations in the transcriptome.
High resolution species assignment of Anopheles mosquitoes using k-mer distances on targeted sequences
Authors: Boddé Marilou , Makunin Alex , Ayala Diego , Bouafa Lemonde , Diabaté Abdoulaye , Ekpo Uwem Friday , Kientega Mahamadi , Goff Gilbert Le , Makanga Boris K. , Ngangue Marc F. , Omitola Olaitan Olamide , Rahola Nil , Tripet Frederic , Durbin Richard , Lawniczak Mara K. N.
Published: March 2022

The ANOSPP amplicon panel is a genus-wide targeted sequencing panel to facilitate large-scale monitoring of Anopheles species diversity. Combining information from the 62 nuclear amplicons present in the ANOSPP panel allows for a more nuanced species assignment than single gene (e.g. COI) barcoding, which is desirable in the light of permeable species boundaries. Here, we present NNoVAE, a method using Nearest Neighbours (NN) and Variational Autoencoders (VAE), which we apply to k-mers resulting from the ANOSPP amplicon sequences in order to hierarchically assign species identity. The NN step assigns a sample to a species-group by comparing the k-mers arising from each haplotype’s amplicon sequence to a reference database. The VAE step is required to distinguish between closely related species, and also has sufficient resolution to reveal population structure within species. In tests on independent samples with over 80% amplicon coverage, NNoVAE correctly classifies to species level 98% of samples within the An. gambiae complex and 89% of samples outside the complex. We apply NNoVAE to over two thousand new samples from Burkina Faso and Gabon, identifying unexpected species in Gabon. NNoVAE presents an approach that may be of value to other targeted sequencing panels, and is a method that will be used to survey Anopheles species diversity and Plasmodium transmission patterns through space and time on a large scale, with plans to analyse half a million mosquitoes in the next five years.
Microglial amyloid beta clearance is driven by PIEZO1 channels
Authors: Konttinen Henna , Sitnikova Valeria , Ishchenko Yevheniia , Shakirzyanova Anastasia , Giudice Luca , Ugidos Irene F , Gómez-Budia Mireia , Korvenlaita Nea , Ohtonen Sohvi , Belaya Irina , Fazaludeen Feroze , Mikhailov Nikita , Gotkiewicz Maria , Ketola Kirsi , Lehtonen Šárka , Koistinaho Jari , Kanninen Katja M , Hernández Damian , Pébay Alice , Giugno Rosalba , Korhonen Paula , Giniatullin Rashid , Malm Tarja
Published: March 2022

Background

Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer’s disease (AD), toxic amyloid beta (Aβ) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aβ. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia have been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored.

Methods

Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC) -derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aβ pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations.

Results

We show that PIEZO1 orchestrates Aβ clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aβ inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aβ clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single cell datasets.

Conclusion

These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aβ burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

GRAPHICAL ABSTRACT
Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages
Authors: Putzeys Leena , Boon Maarten , Lammens Eveline-Marie , Kuznedelov Konstantin , Severinov Konstantin , Lavigne Rob
Published: March 2022

RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5’ and 3’ boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. To this end, we developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in depth transcription studies for microbial viruses or bacteria in general.
Zhx2 is a candidate gene underlying oxymorphone metabolite brain concentration associated with state-dependent oxycodone reward
Authors: Beierle Jacob A. , Yao Emily J. , Goldstein Stanley I. , Lynch William B. , Scotellaro Julia L. , Sena Katherine D. , Wong Alyssa L. , Linnertz Colton A , Averin Olga , Moody David E. , Reilly Christopher A. , Peltz Gary , Emili Andrew , Ferris Martin T. , Bryant Camron D.
Published: March 2022

Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared to the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared to BALB/cByJ mice. Oxymorphone is a highly potent full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identifiedZhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of CYP enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validateZhx2and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains.

Significance Statement

Our findings show genetic variation can result in sex-specific alterations in whole brain concentrations of bioactive opioid metabolites following oxycodone administration, and reinforces the need for sex as a biological factor in pharmacogenomic studies. The co-occurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward which has implications for cue-induced relapse of drug-seeking behavior.
If this title is funny, will you cite me? Citation impacts of humour and other features of article titles in ecology and evolution
Authors: Heard Stephen B. , Cull Chloe A. , White Easton R.
Published: March 2022

Titles of scientific papers pay a key role in their discovery, and “good” titles engage and recruit readers. A particularly interesting aspect of title construction is the use of humour, but little is known about whether funny titles boost or limit readership and citation of papers. We used a panel of volunteer scorers to assess title humour for 2,439 papers in ecology and evolution, and measured associations between humour scores and subsequent citation (both self-citation and citation by others). Papers with funnier titles were cited less often, but this appears to result from a confound with paper importance. Self-citation data suggest that authors give funnier titles to papers they consider less important. After correction for this confound, papers with funny titles have significantly higher citation rates, suggesting that humour recruits readers. We also examined associations between citation rates and several other features of titles. Inclusion of acronyms and taxonomic names was associated with lower citation rates, while assertive-statement phrasing and presence of colons, question marks, and political regions were associated with somewhat higher citation rates. Title length had no effect on citation. Our results suggest that scientists can use creativity with titles without having their work condemned to obscurity.
A microfluidic-based model of nociceptor sensitization reveals a direct activation of sensory axons by prostaglandin E2
Authors: Kimourtzis Georgios , Rangwani Natasha , Jenkins Bethan J. , McNaughton Peter A. , Raouf Ramin
Published: March 2022

Prostaglandin E2 (PGE2) is one of the major contributors to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of the nociceptive axons is incompletely understood. We used a microfluidic cell culture platform to investigate the changes in responsiveness of sensory axons following treatment with PGE2. We show that the application of PGE2 to fluidically isolated axons leads to sensitization of their responses to depolarising stimuli, and the inclusion of zatebradine, a blocker of HCN channels, blocks this enhancement. However, unexpectedly, we also found that the application of PGE2 to the axons elicited a direct and persistent spiking activity in the sensory neurons. We demonstrate that this persistent activity is due to a direct depolarization of axons by PGE2, which is inhibited by Nav1.8 sodium channel blockers but is mainly refractory to Nav1.7 channel blockade. Both the persistent activity and the membrane depolarization in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Our data indicate that PGE2/EP4/cAMP pathway culminates in a sustained depolarization in the sensory axons, leading to the generation of action potentials propagating to the soma. PGE2 therefore, not only mediates nociceptor sensitization but can directly elicit discharges in nociceptive axons, hence redefining its role as a pain mediator in inflammatory conditions.

One Sentence Summary

Prostaglandin E2 can depolarise nociceptive axons in the absence of any noxious stimuli leading to a sustained activation of pain sensing neurons.
Condensin positioning at telomeres by shelterin proteins drives sister-telomere disjunction in anaphase
Authors: Colin Léonard , Reyes Céline , Berthezene Julien , Maestroni Laetitia , Modolo Laurent , Toselli Esther , Chanard Nicolas , Schaak Stephane , Cuvier Olivier , Gachet Yannick , Coulon Stéphane , Bernard Pascal , Tournier Sylvie
Published: March 2022

The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome-remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosome arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis.
Increased male investment in sperm competition results in reduced maintenance of gametes
Authors: Koppik Mareike , Baur Julian , Berger David
Published: March 2022

Male animals often show higher mutation rates than their female conspecifics. A hypothesis for this male-bias is that competition over fertilization of female gametes leads to increased male investment into reproduction at the expense of maintenance and repair, resulting in a trade-off between male success in sperm competition and offspring quality. Here, we provide evidence for this hypothesis by harnessing the power of experimental evolution to study effects of sexual selection on the male germline in the seed beetleCallosobruchus maculatus. We first show that 50 generations of evolution under strong sexual selection, coupled with experimental removal of natural selection resulted in males that are more successful in sperm competition. We then show that these males produce progeny of lower quality if engaging in socio-sexual interactions prior to being challenged to surveil and repair experimentally induced damage in their germline, and that the presence of male competitors alone can be enough to elicit this response. We identify 18 candidate genes that showed differential expression in response to the induced germline damage, with several of these previously implicated in processes associated with DNA repair and cellular maintenance. These genes also showed significant expression changes across socio-sexual treatments of fathers and predicted the reduction in quality of their offspring, with expression of one gene also being strongly correlated to male sperm competition success. Sex differences in expression of the same 18 genes indicate a substantially higher female investment in germline maintenance. While more work is needed to detail the exact molecular underpinnings of our results, our findings provide rare experimental evidence for a trade-off between male success in sperm competition and germline maintenance. This suggests that sex-differences in the relative strengths of sexual and natural selection are causally linked to male mutation bias. The tenet advocated here, that the choices of an individual can affect plasticity of its germline and the resulting genetic quality of subsequent generations, has several interesting implications for mate choice processes.
A role of Lck annular lipids in the steady upkeep of active Lck in T cells
Authors: Porciello Nicla , Cipria Deborah , Masi Giulia , Lanz Anna-Lisa , Milanetti Edoardo , Grottesi Alessandro , Howie Duncan , Cobbold Steve P. , Schermelleh Lothar , He Hai-Tao , D’Abramo Marco , Destainville Nicolas , Acuto Oreste , Nika Konstantina
Published: March 2022

Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) can be modulated by annular lipids sheathing their hydrophobic moiety. Here, we present evidence for this prediction in a natural membrane by investigating the mechanism that maintains steady amount of active isoform of Lck kinase (LckA) by Lck trans-autophosphorylation offset by the phosphatase CD45. We gauged experimental suitability by quantitation of CD45 and LckAsubcellular localisation, LckAgeneration as a function of Lck and pharmacological perturbation. Steady LckAwas challenged by swapping Lck membrane anchor with structurally divergent ones expected to substantially modify Lck annular lipids, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45, respectively. The data showed only small alteration of LckA, except for CD45 hydrophobic anchor that thwarted LckA, due to excessive lateral proximity to CD45. The data are best explained by annular lipids facilitating or penalising IMPs’ lateral proximity, hence modulating IMPs protein-protein functional interactions. Our findings can contribute to improve the understanding of biomembranes’ organisation.
Strategies to Identify and Edit Improvements in Synthetic Genome Segments Episomally
Authors: Rudolph Alexandra , Nyerges Akos , Chiappino-Pepe Anush , Landon Matthieu , Baas-Thomas Maximilien , Church George
Published: March 2022

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments on low copy number vectors. However, DNA synthesis and amplification have the potential to impose mutations on the contained DNA segments that can in turn reduce or prevent viability of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to ‘recoding’ an Escherichia coli strain to swap out seven codons to synonymous alternatives genome-wide. Ten 44,402 to 47,179 bp de novo synthesized BAC-contained DNA segments from the recoded strain were unable to complement deletion of the corresponding 33 to 61 wild type genes using a single antibiotic resistance marker. Next-Generation Sequencing was used to identify 1-7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the BAC-contained recoded segment when both the recoded and wild type copies of the mutated genes had to exist by necessity during the repair process. This strategy could be adapted to mutation identification and repair for other large-scale genome engineering projects, or for incorporation of small genetic engineering sites for quick protocol adjustments.
MINSTED nanoscopy enters the Ångström localization range
Authors: Weber Michael , von der Emde Henrik , Leutenegger Marcel , Gunkel Philip , Cordes Volker C. , Sambandan Sivakumar , Khan Taukeer A. , Keller-Findeisen Jan , Hell Stefan W.
Published: March 2022

We report all-optical, room-temperature localization of fluorophores with precision in the Ångström range. These precisions are attained in a STED microscope, by encircling the fluorophore with the low-intensity edge of the STED donut beam, while constantly increasing the absolute donut power. Individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Since our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up entirely new areas of application in the study of macromolecular complexes in cells.
Hepatocyte-specific loss of LAP2α protects against diet-induced hepatic steatosis and steatohepatitis in male mice
Authors: Upadhyay Kapil K. , Choi Eun-Young K. , Foisner Roland , Omary M. Bishr , Brady Graham F.
Published: March 2022

There is increasing evidence for the importance of the nuclear envelope in lipid metabolism, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Human mutations in LMNA, encoding A-type nuclear lamins, cause early-onset insulin resistance and NASH, while hepatocyte-specific deletion of Lmna predisposes to NASH with fibrosis in male mice. Given that variants in the gene encoding LAP2α, a nuclear protein that regulates lamin A/C, were previously identified in patients with NAFLD, we sought to determine the role of LAP2α in NAFLD using a mouse genetic model. Hepatocyte-specific Lap2a-knockout (Lap2α(ΔHep)) mice and littermate controls were fed normal chow or high-fat diet (HFD) for 8 weeks or 6 months. In contrast to what was observed with hepatocyte-specific Lmna deletion, male Lap2a(ΔHep) mice showed no increase in hepatic steatosis or NASH compared to controls. Rather, Lap2a(ΔHep) mice demonstrated reduced hepatic steatosis, particularly after long-term HFD, with decreased susceptibility to diet-induced NASH. Accordingly, whereas pro-steatotic genes Cidea, Mogat1, and Cd36 were upregulated in Lmnα-KO mice, they were downregulated in Lap2α(ΔHep) mice, and there was a trend toward decreases in pro-inflammatory and pro-fibrotic genes. These data indicate that hepatocyte-specific Lap2a deletion protects against hepatic steatosis and NASH in mice; therefore, LAP2α might represent a potential therapeutic target in human NASH.

Brief Summary

Loss of LAP2α in mouse hepatocytes protected against diet-induced hepatic steatosis and NASH.
Alpha-1 Antitrypsin Limits Neutrophil Extracellular Trap Disruption of Airway Epithelial Barrier Function
Authors: Hudock KM , Collins MS , Imbrogno M , Kramer EL , Brewington JJ , Ziady A , Zhang N , Snowball J , Xu Y , Carey BC , Horio Y , O’Grady SM , Kopras EJ , Meeker J , Morgan H , Ostmann AJ , Skala E , Siefert ME , Na CL , Gollomp K , Mangalmurti N , Trapnell BC , Clancy JP
Published: March 2022

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.
Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase-dependent
Authors: Kim Seonghoon , Hyeon Changbong
Published: March 2022

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance (EPR) spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that for DPPC membrane whose gel-to-fluid lipid phase transition occurs at 314 K, TEMPO is stabilized above (below) the bilayer interface if the membrane is in the gel (fluid) phase. For bilayers made of unsaturated lipids, DOPC and POPC, which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.
Characterizing dry mass and volume changes in human multiple myeloma cells upon treatment with proteotoxic and genotoxic drugs
Authors: Liu Xili , Moscvin Maria , Oh Seungeun , Chen Tianzeng , Choi Wonshik , Evans Benjamin , Rowell Sean M. , Nadeem Omar , Mo Clifton C. , Sperling Adam S. , Anderson Kenneth C. , Yaqoob Zahid , Bianchi Giada , Sung Yongjin
Published: March 2022

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients, however limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally-enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 hour for sensitive cells and 4 hours for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.
One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant
Authors: Buck Kevin M. , Roberts David S. , Aballo Timothy J. , Inman David R. , Jin Song , Ponik Suzanne , Brown Kyle A. , Ge Ying
Published: March 2022

Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content of exosomes but the current methods require laborious and time-consuming multi-step sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, for rapid and effective exosomal lysis, protein extraction, and digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3,466 proteins and quantified 2,288 proteins using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. 3,166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.
SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma
Authors: Tonelli Claudia , Yordanov Georgi N. , Hao Yuan , Deschênes Astrid , Klingbeil Olaf , Ting Hsiu-Chi , Brosnan Erin , Doshi Abishek , Park Youngkyu , Vakoc Christopher R. , Preall Jonathan , Tuveson David A.
Published: March 2022

Pancreatic ductal adenocarcinoma (PDA) samples reveal extensive cellular heterogeneity. Using single-cell RNA sequencing, we uncover multiple tumor cell populations distinguished by their differentiation state and associated with different stages of tumor progression in a mouse model of PDA. We identify Spdef as a factor required for tumorigenesis in pancreatic cancer cells of epithelial and mucinous nature. By comparative analysis of cell differentiation states in mice and humans, we find that the Spdef program is highly expressed by human PDAs of the classical subtype. Mouse and human PDA cells expressing elevated levels of Spdef are dependent upon this transcription factor for tumor progression in vivo. The tumor-promoting function of Spdef is recapitulated by two Spdef target genes that regulate protein folding and endoplasmic reticulum activity, Agr2 and Ern2/Ire1β. These findings offer insights into the factors controlling differentiation states in PDA and identify new vulnerabilities in the most common subtype of pancreatic cancer.
Activation of the chemokine receptor CCR1 and preferential recruitment of Gαi suppress RSV replication: implications for developing novel RSV treatment strategies
Authors: Li Jiao , Xue Ling , Wang Jiachao , Meng Aihong , Qiao Jiajun , Li Miao , Wang Xiuli , Meng Lingtong , Ning Jingyuan , Gao Xue , Li Wenjian , Ma Cuiqing , Wei Lin
Published: March 2022

Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in non-immune cells is rarely clear. In this study, we detect that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo/in vitro, viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction or viral adhesion or entry inhibition; in contrast, it was related to the preferential recruitment and activation of adaptor Gαi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation, explicitly, phosphorylated (p)-STAT3 was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of non-immune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction.
Chloroplast protein import determines plant proteostasis and retrograde signaling
Authors: Llamas Ernesto , Koyuncu Seda , Lee Hyun Ju , Gutierrez-Garcia Ricardo , Dunken Nick , Charura Nyasha , Torres-Montilla Salvador , Schlimgen Elena , Pulido Pablo , Rodriguez-Concepcion Manuel , Zuccaro Alga , Vilchez David
Published: March 2022

Proteins containing polyglutamine (polyQ) repeats are prone to aggregation and can lead to distinct human pathologies. For instance, Huntington’s disease is caused by an abnormal expansion of the polyQ stretch (> Q35) of Huntingtin (HTT) protein. However, plants express hundreds of proteins containing polyQ regions, but no pathologies arising from these factors have been reported to date. Here, we ask how plants maintain the proteostasis of polyQ-containing proteins, which are intrinsically enriched in the plant proteomes. To this end, we overexpressed an aggregation-prone fragment of human HTT (Q69) in plant cells. In contrast to invertebrate and mammalian transgenic models, we find that Arabidopsis thaliana plants suppress Q69 aggregation. This elevated proteostasis ability is mediated through the import and degradation of Q69 in chloroplasts. Conversely, inhibition of chloroplast protein import either genetically or pharmacologically reduces the capacity of plant cells to prevent Q69 aggregation. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Notably, expression of synthetic Arabidopsis SPP is sufficient to suppress aggregation of polyQ-expanded HTT in human cells. Beyond ectopically expressed Q69-HTT, endogenous polyQ-containing proteins also aggregate in Arabidopsis upon inhibition of chloroplast import. Among them, the plastid casein kinase 2 (pCK2), which contains a polyQ region next to the chloroplast targeting sequence motif, can also be localized into the nucleus. Upon inhibition of chloroplast import, pCK2 accumulates at higher levels in the nucleus and forms diamond-shaped amyloid-like fibrils surrounding the chloroplasts. These results indicate that the differential conformation and redistribution of pCK2 to the nucleus depends on chloroplast import efficiency, providing a role of polyQ repeats in chloroplast to nucleus communication (i.e. retrograde signaling). Together, our findings establish chloroplast protein import and proteases as determinants of polyQ proteostasis, with important implications for plant biology that can also lead to therapeutic approaches for human diseases that involve protein aggregation.
Configurable Compartmentation Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems
Authors: Li Luyao , Zhang Rong , Tian Xintong , Li Ting , Pu Bingchun , Ma Conghui , Ba Fang , Xiong Chenwei , Shi Yunfeng , Li Jian , Keasling Jay , Zhang Jingwei , Liu Yifan
Published: March 2022

The compartmentalized and communicative nature of biological cells contributes to the complexity and endurance of living organisms. Current in vitro compartmentalization systems such as droplet emulsions reproduce the compartmentalization property of cells yet fail to recapture the configurability of cellular communication with the environment. To mimic biological cells a step further and expand the capabilities of in vitro compartmentalization, we present here a general strategy that inherits the passive transport phenomenon of biology. The strategy incorporates layered, micrometer-sized, hydrogel-based compartments featuring configurability in composition, functionality, and selective permeability of biomolecules. We demonstrated the unique advantage of our strategy in two scenarios of synthetic biology. First, a compartmentalized cell-free protein synthesis system was reconstituted that could support multiple rounds of reactions. Second, we constructed living bacteria-based biosensors in the hydrogel compartments, which could achieve long-lasting functioning with markedly enhanced fitness in complex environments. Looking forward, our strategy should be widely applicable for constructing complex, robust, and sustained in vitro synthetic molecular and cellular systems, paving the way for their practical applications.