bluuebunny/crossref_metadata_2025 huggingface dataset:
Systems biology analysis identifies TCF7L1 as a key regulator of metastasis in Ewing sarcoma
Authors: Cidre-Aranaz Florencia , Li Jing , Hölting Tilman L. B. , Orth Martin F. , Imle Roland , Kutschmann Stefanie , Ammirati Giulia , Ceranski Katharina , Carreño-Gonzalez Martha Julia , Kasan Merve , Marchetto Aruna , Funk Cornelius M. , Bestvater Felix , Bersini Simone , Arrigoni Chiara , Moretti Matteo , Romero-Pérez Laura , Banito Ana , Ohmura Shunya , Musa Julian , Kirchner Thomas , Knott Maximilian M. L. , Grünewald Thomas G. P.
Published: February 2021

Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (<30%) for patients with metastatic disease at diagnosis (∼25% of cases). Hence, deciphering the underlying mechanisms of metastasis is imperative. EwS tumors are characterized by a remarkably ‘silent’ genome with a single driver mutation generating an oncogenic fusion transcription factor (EWSR1-ETS). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated.

Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 (TCF7L1, aliasTCF3) as a prognostically-relevant andEWSR1-ETSsuppressed determinant of metastasis in EwS. We demonstrated that conditionalTCF7L1re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assaysin vitro, and tumorigenesisin vivomediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopicin vivomodels, lowTCF7L1expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients.

Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.
Mechanically Sensitive HSF1 is a Key Regulator of Left-Right Symmetry Breaking in Zebrafish Embryos
Authors: Du Jing , Li Shu-Kai , Guan Liu-Yuan , Guo Zheng , Yin Jiang-Fan , Gao Li , Kawanishi Toru , Shimada Atsuko , Zhang Qiu-Ping , Zheng Li-Sha , Liu Yi-Yao , Feng Xi-Qiao , Chen Dong-Yan , Takeda Hiroyuki , Fan Yu-Bo
Published: February 2021

The left-right symmetry breaking of vertebrate embryos requires fluid flow (called nodal flow in zebrafish). However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. In this paper, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kuppfer’s vesicle at the early stage of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significantsitus inversusand disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 could be immediately activated by fluid shear stress. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and the Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stressin vitroandin vivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation triggered by mechanical clues during embryonic development and other physiological and pathological transformations.
Dissociable oscillatory networks support gain and loss processing in human orbitofrontal cortex
Authors: Saez Ignacio , Lin Jack , Chang Edward , Parvizi Josef , Knight Robert T. , Hsu Ming
Published: February 2021

Human neuroimaging and animal studies have linked neural activity in orbitofrontal cortex (OFC) to valuation of positive and negative outcomes. Additional evidence shows that neural oscillations, representing the coordinated activity of neuronal ensembles, support information processing in both animal and human prefrontal regions. However, the role of OFC neural oscillations in reward-processing in humans remains unknown, partly due to the difficulty of recording oscillatory neural activity from deep brain regions. Here, we examined the role of OFC neural oscillations (<30Hz) in reward processing by combining intracranial OFC recordings with a gambling task in which patients made economic decisions under uncertainty. Our results show that power in different oscillatory bands are associated with distinct components of reward evaluation. Specifically, we observed a double dissociation, with a selective theta band oscillation increase in response to monetary gains and a beta band increase in response to losses. These effects were interleaved across OFC in overlapping networks and were accompanied by increases in oscillatory coherence between OFC electrode sites in theta and beta band during gain and loss processing, respectively. These results provide evidence that gain and loss processing in human OFC are supported by distinct low-frequency oscillations in networks, and provide evidence that participating neuronal ensembles are organized functionally through oscillatory coherence, rather than local anatomical segregation.
Can we fish on stocks that need rebuilding? Illustrating the trade-offs between stock conservation and fisheries considerations
Authors: Trijoulet Vanessa , Berg Casper W. , Sparrevohn Claus R. , Nielsen Anders , Pastoors Martin A. , Mosegaard Henrik
Published: February 2021

In the Northeast Atlantic, advice for many fish stocks follows the ICES MSY approach, where a zero catch will be recommended if the stock is below its limit reference point,Blim, and cannot rebuild in the short-term. How-ever, zero catch advice are rarely implemented by managers. This study used medium-term stochastic forecasts with harvest control rules (HCRs) to investigate the consequences of allowing reduced fishing belowBlim. We applied the method to western Baltic herring and North Sea cod, two contrasting species currently estimated belowBlim. We show that the minimum rebuilding probability of 95% required by the MSY approach could be impossible to reach in the short-to medium-term. When this is the case, a lower probability may need to be considered instead in the short-term. Recruitment is the largest source of uncertainty in stock response to management, and can exceed differences between HCRs. Reference points should be estimated in accordance with current recruitment levels if they are to be used for short-term advice or as realistic rebuilding targets. For both stocks, it is possible to keep fishing at reduced levels for similar cumulative catch, SSB and risk on the stock in the medium-term compared to no catch belowBlim. Medium-term trade-offs between stock conservation and fisheries considerations may be needed when fishery closure cannot be implemented in practice.
Metabolomics of aging in primary fibroblasts from small and large breed dogs
Authors: Brookes Paul S. , Jimenez Ana G.
Published: February 2021

Among several animal groups (eutherian mammals, birds, reptiles) lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in co-enzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small versus large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.
Unsupervised learning of haptic material properties
Authors: Metzger Anna , Toscani Matteo
Published: February 2021

When touching the surface of an object, its spatial structure translates into a vibration on the skin. The perceptual system evolved to translate this pattern into a representation that allows to distinguish between different materials. Here we show that perceptual haptic representation of materials emerges from efficient encoding of vibratory patterns elicited by the interaction with materials. We trained a deep neural network with unsupervised learning (Autoencoder) to reconstruct vibratory patterns elicited by human haptic exploration of different materials. The learned compressed representation (i.e. latent space) allows for classification of material categories (i.e. plastic, stone, wood, fabric, leather/wool, paper, and metal). More importantly, distances between these categories in the latent space resemble perceptual distances, suggesting a similar coding. We could further show, that the temporal tuning of the emergent latent dimensions is similar to properties of human tactile receptors.
Versatile cell-based assay for measuring DNA alkylation damage and its repair
Authors: Li Yong , Mao Peng , Basenko Evelina Y. , Lewis Zachary , Smerdon Michael J. , Czaja Wioletta
Published: February 2021

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.
Proanthocyanidin-enriched cranberry extract induces resilient bacterial community dynamics in a gnotobiotic mouse model
Authors: Neto Catherine C. , Mortzfeld Benedikt M. , Turbitt John R. , Bhattarai Shakti K. , Yeliseyev Vladimir , DiBenedetto Nicholas , Bry Lynn , Bucci Vanni
Published: February 2021

Cranberry consumption has numerous health benefits, with experimental reports showing its anti-inflammatory and anti-tumor properties. Importantly, microbiome research has demonstrated that the gastrointestinal bacterial community modulates host immunity, raising the question whether the cranberry-derived effect may be related to its ability to modulate the microbiome. Only a few studies have investigated the effect of cranberry products on the microbiome to date. Especially because cranberry is rich in dietary fibers, we do not know the extent of microbiome modulation that is caused solely by polyphenols, particularly proanthocyanidins (PACs). Since previous work has only focused on the long-term effects of cranberry extracts, in this study we investigated the effect of a water-soluble, polyphenol-rich cranberry juice extract (CJE) on the short-term dynamics of human-derived bacterial community in a gnotobiotic mouse model. CJE characterization revealed a high enrichment in PACs (57% PACs), the highest ever utilized in a microbiome study. In a 37-day experiment with a 10-day CJE intervention and 14-day recovery time, we profiled the microbiota via 16 rDNA sequencing and applied diverse time-series analytics methods to identify individual bacterial responses. We show that daily administration of CJE induces distinct dynamical patterns in bacterial abundances during and after treatment before recovering resiliently to pre-treatment levels. Specifically, we observed an increase of the immunomodulatory mucin degradingAkkermansia muciniphilaafter treatment, suggesting intestinal mucus accumulation due to CJE. Interestingly, this expansion coincided with an increase in the abundance of butyrate-producing Clostridia, a group of microbes known to promote numerous adaptive and innate anti-inflammatory phenotypes.
Exploring the interaction network of a synthetic gut bacterial community
Authors: Weiss Anna S. , Burrichter Anna G. , Durai Raj Abilash Chakravarthy , von Strempel Alexandra , Meng Chen , Kleigrewe Karin , Münch Philipp C. , Rössler Luis , Huber Claudia , Eisenreich Wolfgang , Jochum Lara M. , Göing Stephanie , Jung Kirsten , Sanchez Alvaro , Stecher Bärbel
Published: February 2021

A key challenge in microbiome research is to predict functionality from microbial community composition. As central microbiota functions are determined by bacterial community networks it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments, as well as in community batch culture. Metabolomics analysis of spent culture supernatant of individual strains in combination with genome-informed pathway reconstruction provided insights into the metabolic potential of the individual community members. Thereby, we could show that the OMM12interaction network is shaped by both, exploitative and interference competitionin vitro.In particular,Enterococcus faecalisKB1 was identified as important driver of community composition by affecting the abundance of several other consortium members. Together, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12interaction network, which serves as knowledge base for future mechanistic studies.
Local Accumulation of Axonal Mitochondria in the Optic Nerve Glial Lamina Precedes Myelination
Authors: Wilkison Samantha J. , Bright Cora L. , Vancini Ricardo , Song Daniel J. , Bomze Howard M. , Cartoni Romain
Published: February 2021

Mitochondria are essential for neurons and must be optimally distributed along their axon to fulfil local functions. A high density of mitochondria has been observed in retinal ganglion cell (RGC) axons of an unmyelinated region of the optic nerve, called the glial lamina (GL) in mouse (lamina cribrosa in human). In glaucoma, the world’s leading cause of irreversible blindness, the GL is the epicenter of RGC degeneration and is connected to mitochondrial dysfunction. It is generally accepted that the local accumulation of mitochondria in the GL is established due to the higher energy requirement of unmyelinated axons. Here we revisit the connection between mitochondrial positioning and myelin in RGC axons. We show that the high density of mitochondria in the GL is restricted to larger axons and is established before myelination. Thus, contrary to a longstanding belief in the field, the myelination pattern is not responsible for the establishment of the local accumulation of mitochondria in GL axons. Our findings open new research avenues likely critical to understanding the pathophysiology of glaucoma.
Defining kinetic roles of transcriptional activators in the early Drosophila embryo
Authors: Harden Timothy T. , Vincent Ben J. , DePace Angela H.
Published: February 2021

Most animal transcription factors are categorized as activators or repressors without specifying their mechanisms of action. Defining their specific roles is critical for deciphering the logic of transcriptional regulation and predicting the function of regulatory sequences. Here, we define the kinetic roles of three activating transcription factors in the Drosophila embryo—Zelda, Bicoid and Stat92E—by introducing their binding sites into theeven skippedstripe 2 enhancer and measuring transcriptional output with live imaging. We find that these transcription factors act on different subsets of kinetic parameters, and these subsets can change over the course of nuclear cycle (NC) 14. These transcription factors all increase the fraction of active nuclei. Zelda dramatically shortens the time interval between the start of NC 14 and initial activation, and Stat92E increases the duration of active transcription intervals throughout NC 14. Zelda also decreases the time intervals between instances of active transcription early in NC 14, while Stat92E does so later. Different transcription factors therefore play distinct kinetic roles in activating transcription; this has consequences for understanding both regulatory DNA sequences as well as the biochemical function of transcription factors.
The Diameter Factor of Aligned Membranes Facilitates Cutaneous Wound Healing by Promoting Epithelialization in an Immune Way
Authors: Wang Chenbing , Chu Chenyu , Zhao Xiwen , Hu Chen , Liu Li , Li Jidong , Qu Yili , Man Yi
Published: February 2021

Topographical properties (such as pattern and diameter) of biomaterials play important roles in influencing cell activities and manipulating the related immune response during the skin wound healing. We prepared aligned electrospinning membranes with different fiber diameters including 319±100 nm (A300), 588±132 nm (A600) and 1048±130 nm (A1000). The A300 membranes significantly improved cell proliferation and spreading, and facilitated skin wound healing (epithelization and vascularization) with the regeneration of hair follicles. The transcriptome revealed the underlying molecular mechanism that A300 could promote macrophages transformation to reparative phenotypes with highly expressed IL10 and TGFβ, subsequently promoting epidermal cell migration via secreting matrix metalloproteinases 12 (MMP12). All the results indicate the A300 to be a potential candidate for guided skin regeneration applications.

Graphic abstract
Termination of DNA replication at Tus-terbarriers results in under-replication of template DNA
Authors: Jameson Katie H. , Rudolph Christian J. , Hawkins Michelle
Published: February 2021

The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. InEscherichia coli, replication termination, the final stage of the duplication process, is confined to the ‘replication fork trap’ region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomictersites. Termination typically occurs away from Tus-tercomplexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-terand termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-tercomplexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-tercomplexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-terbarriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.
An integrated multi-wavelength SCATTIRSTORM microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes
Authors: Nong Daguan , Haviland Zachary K. , Kuntz Kate Vasquez , Tien Ming , Anderson Charles T. , Hancock William O.
Published: February 2021

We describe a multimodal SCATTIRSTORM microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines Interference Reflection Microscopy (IRM) with multi-wavelength Total Internal Reflectance Fluorescence Microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.
Early functional and cognitive declines measured by auditory evoked cortical potentials in Alzheimer’s disease mice
Authors: Mei Ling , Liu Li-Man , Chen Kaitian , Zhao Hong-Bo
Published: February 2021

Alzheimer’s disease (AD) is characterized with a progressive loss of memory and cognitive decline. However, assessment of AD-associated functional and cognitive changes is still a big challenge. Auditory evoked cortical potential (AECP) is an event-related potential reflecting not only neural activation in the auditory cortex but also cognitive activity in the brain. In this study, we used the subdermal needle electrodes with the same electrode setting as the auditory brainstem response (ABR) recording and recorded AECP in normal aging CBA/CaJ mice and APP/PS1 AD mice. AECP in mice usually appeared three positive peaks, i.e., P1, P2, and P3, and three corresponding negative peaks N1, N2, and N3. In normal aging CBA mice, the early sensory peaks P1, N1, and P2 were reduced as age increased, whereas the later cognitive peaks N2, P3, and N3 were increased or had no changes with aging. Moreover, the latency of P1 peak was increased as age increased, although the latencies of later peaks had significant reduction with aging. In AD mice, peak P1 was significantly reduced in comparison with wild-type (WT) littermates at young ages, proceeding AD phenotype presentation. In particular, the later cognitive peak P3 was diminished after 3 months old, different from normal aging effect. However, the latencies of AECP peaks in AD mice generally had no significant delay or changes with aging. Finally, consistent with AECP changes, the accumulation of amyloid precursor protein (APP) at the auditory cortex was visible in AD mice as early as 2 months old. These data suggest that AECP could serve as an early, noninvasive, and objective biomarker for detecting AD and AD-related dementia.
Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants
Authors: Wang Lingshu , Zhou Tongqing , Zhang Yi , Yang Eun Sung , Schramm Chaim A. , Shi Wei , Pegu Amarendra , Oloninyi Olamide K. , Ransier Amy , Darko Samuel , Narpala Sandeep R. , Hatcher Christian , Martinez David R. , Tsybovsky Yaroslav , Phung Emily , Abiona Olubukola M. , Cale Evan M. , Chang Lauren A. , Corbett Kizzmekia S. , DiPiazza Anthony T. , Gordon Ingelise J. , Leung Kwanyee , Liu Tracy , Mason Rosemarie D. , Nazzari Alexandra , Novik Laura , Olia Adam S. , Doria-Rose Nicole A. , Stephens Tyler , Stringham Christopher D. , Talana Chloe Adrienna , Teng I-Ting , Wagner Danielle , Widge Alicia T. , Zhang Baoshan , Roederer Mario , Ledgerwood Julie E. , Ruckwardt Tracy J. , Gaudinski Martin R. , Baric Ralph S. , Graham Barney S. , McDermott Adrian B. , Douek Daniel C. , Kwong Peter D. , Mascola John R , Sullivan Nancy J. , Misasi John
Published: February 2021

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50<0.0006 to 0.0102 μg/mL; IC80 < 0.0006 to 0.0251 μg/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease thein vitrogeneration of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.

One Sentence Summary

Ultrapotent antibodies from convalescent donors neutralize and mitigate resistance of SARS-CoV-2 variants of concern.
Different underlying mechanisms for high and low arousal in probabilistic learning in humans
Authors: Ciria Luis F. , Suárez-Pinilla Marta , Williams Alex G. , Jagannathan Sridhar R. , Sanabria Daniel , Bekinschtein Tristán A.
Published: February 2021

Humans are uniquely capable of adapting to highly changing environments by updating relevant information and adjusting ongoing behaviour accordingly. Here we show how this ability —termed cognitive flexibility— is differentially modulated by high and low arousal fluctuations. We implemented a probabilistic reversal learning paradigm in healthy participants as they transitioned towards sleep or physical extenuation. The results revealed, in line with our pre-registered hypotheses, that low arousal leads to diminished behavioural performance through increased decision volatility, while performance decline under high arousal was attributed to increased perseverative behaviour. These findings provide evidence for distinct patterns of maladaptive decision-making on each side of the arousal inverted u-shaped curve, differentially affecting participants’ ability to generate stable evidence-based strategies, and introduces wake-sleep and physical exercise transitions as complementary experimental models for investigating neural and cognitive dynamics.
H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeller function
Authors: Li Jian , Bergmann Lena , de Almeida Andreia Rafael , Webb Kimberly M. , Gogol Madelaine M. , Voigt Philipp , Liu Yingfang , Liang Huanhuan , Smolle Michaela M.
Published: February 2021

The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promote the interaction of full-length Ioc4 with nucleosomesin vitroand they are necessary for its recruitment to gene bodiesin vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodelling by Isw1b and the maintenance of ordered chromatinin vivo, thereby preventing the production of non-coding RNAs.
Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus
Authors: Vaidyanathan Sriram , Baik Ron , Chen Lu , Bravo Dawn T. , Suarez Carlos J. , Abazari Shayda M. , Salahudeen Ameen A. , Dudek Amanda M. , Teran Christopher A. , Davis Timothy H. , Lee Ciaran M. , Bao Gang , Randell Scott H. , Artandi Steven E. , Wine Jeffrey J. , Kuo Calvin J. , Desai Tushar J. , Nayak Jayakar V. , Sellers Zachary M. , Porteus Matthew H.
Published: February 2021

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.
Quantitative mapping of dense microtubule arrays in mammalian neurons
Authors: Katrukha Eugene A. , Jurriens Daphne , Pastene Desiree Salas , Kapitein Lukas C.
Published: February 2021

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network. This revealed that in dendrites stable, acetylated microtubules are enriched in the core of the dendritic shaft, while dynamic, tyrosinated microtubules enrich near the plasma membrane, thus forming a shell around the stable microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ∼20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport.
Activity-induced fluidization of arrested coalescence in fusion of cellular aggregates
Authors: Ongenae Steven , Cuvelier Maxim , Vangheel Jef , Ramon Herman , Smeets Bart
Published: February 2021

At long time scales, tissue spheroids may flow or appear solid depending on their capacity to reorganize their internal structure. Understanding the relationship between intrinsic mechanical properties at the single cell level, and the tissue spheroids dynamics at the long-time scale is key for artificial tissue constructs, which are assembled from multiple tissue spheroids that over time fuse to form coherent structures. The dynamics of this fusion process are frequently analyzed in the framework of liquid theory, wherein the time scale of coalescence of two droplets is governed by its radius, viscosity and surface tension. In this work, we extend this framework to glassy or jammed cell behavior which can be observed in spheroid fusion. Using simulations of an individual-cell based model, we demonstrate how the spheroid fusion process can be steered from liquid to arrested by varying active cell motility and repulsive energy as established by cortical tension. The divergence of visco-elastic relaxation times indicates glassy relaxation near the transition towards arrested coalescence. Finally, we investigate the role of cell growth in spheroid fusion dynamics. We show that the presence of cell division introduces plasticity in the material and thereby increases coalescence during fusion.
A new parallel high-pressure packing system enables rapid multiplexed production of capillary columns
Authors: Müller-Reif Johannes B. , Hansen Fynn M. , Schweizer Lisa , Treit Peter V. , Geyer Philipp E. , Mann Matthias
Published: February 2021

Reversed-phase high performance liquid chromatography (HPLC) is the most commonly applied peptide separation technique in mass spectrometry (MS)-based proteomics. Particle-packed capillary columns are predominantly used in nano-flow HPLC systems. Despite being the broadly applied standard for many years capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column production with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as driving fluid, our system replaces the traditional setup of helium pressured packing bombs. By using 10x multiplexing, we have reduced the production time to just under 2 minutes for several 50 cm columns with 1.9 µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters (ID) and length packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station.

One sentence summary

A newly constructed parallel high-pressure packing system enables the rapid multiplexed production of capillary columns.

Abstract Figure
Stability of ligand-induced protein conformation influences affinity in maltose-binding protein
Authors: van den Noort Marco , de Boer Marijn , Poolman Bert
Published: February 2021

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) fromEscherichia coli. We used singlemolecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.
Comparative infectivity and pathogenesis of emerging SARS-CoV-2 variants in Syrian hamsters
Authors: Abdelnabi Rana , Boudewijns Robbert , Foo Caroline S. , Seldeslachts Laura , Sanchez-Felipe Lorena , Zhang Xin , Delang Leen , Maes Piet , Kaptein Suzanne J. F. , Weynand Birgit , Velde Greetje Vande , Neyts Johan , Dallmeier Kai
Published: February 2021

Within one year after its emergence, more than 108 million people contracted SARS-CoV-2 and almost 2.4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1), variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics and new spikes in excess mortality. We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 lineages in Syrian golden hamsters to assess their relative infectivity and pathogenicity in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Overall, we established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC.
Clinical response to dabrafenib plus trametinib in a pediatric ganglioglioma with BRAF p.T599dup mutation
Authors: Miller Katherine E. , Schieffer Kathleen M. , Grischow Olivia , Rodriguez Diana P. , Cottrell Catherine E. , Leonard Jeffrey R. , Finlay Jonathan L. , Mardis Elaine R.
Published: February 2021

In this follow-up report, we present updated information regarding a previously reported pediatric patient with a World Health Organization grade I ganglioglioma harboring a BRAF p.T599dup mutation (Cold Spring Harb Mol Case Stud 4: a002618). This patient, based on our initial finding, is receiving combination targeted therapy with a selective BRAF inhibitor (dabrafenib) plus MEK inhibitor (trametinib). The combination therapy was started after the patient experienced progressive tumor growth and worsening neurological symptoms, including visual changes, headaches, and peripheral neuropathy, despite 9 months of treatment with adjuvant chemotherapy (vinblastine). The patient has been receiving dabrafenib plus trametinib for 15 months and continues to have stable disease as well as improved neurological symptoms. Although combinatorial therapy targeting BRAF and MEK using dabrafenib and trametinib, respectively, is indicated for tumors harboring a BRAF p.V600E/K mutation, our report demonstrates efficacy of this combination in a non-V600E BRAF-mutated tumor. The identification of BRAF alterations may assist clinicians in determining alternative targeted treatment strategies, especially considering the paucity of effective treatments for primary brain tumors and the poor prognosis associated with many central nervous system (CNS) diagnoses. Additional case studies or larger cohort reports will continue to clarify the efficacy of BRAF and/or MEK inhibitors in patients whose tumors harbor a BRAF alteration.
Syntheses and structures of two benzoyl amides: 2-chloro-4-ethoxy-3,5-dimethoxy-N-(3-oxocyclohex-1-en-1-yl)benzamide and 2-chloro-N-(5,5-dimethyl-3-oxocyclohex-1-en-1-yl)-4-ethoxy-3,5-dimethoxybenzamide
Authors: Anderson Alan J. , Butcher Ray J. , Ollie Edward
Published: February 2021

The first title benzoyl amide, C17H20ClNO5 (3a), crystallizes in the monoclinic space group P21/c with Z = 4 and the second, C19H24ClNO5 (3b), also crystallizes in P21/c with Z = 8 (Z′ = 2), thus there are two independent molecules in the asymmetric unit. In 3a, the phenyl ring makes a dihedral angle of 50.8 (3)° with the amide moiety with the C=O group on the same side of the molecule as the C—Cl group. One methoxy group is almost in the plane of the benzene ring, while the ethoxy and other methoxy substituent are arranged on opposite sides of the ring with the ethoxy group occupying the same side of the ring as the C=O group in the amide moiety. For one of the two molecules in 3b, both the amide and 5,5-dimethyl-3-oxocyclohex-1-en-1-yl moieties are disordered over two sets of sites with occupancies of 0.551 (2)/0.449 (2) with the major difference between the two conformers being due to the conformation adopted by the cyclohex-2-en-1-one ring. The three molecules in 3b (i.e., the undisordered molecule and the two disorder components) differ in the arrangement of the subsituents on the phenyl ring and the conformation adopted by their 5,5-dimethyl-3-oxocyclohex-1-en-1-yl moieties. In the crystal of 3a, N—H...O hydrogen bonds link the molecules into a zigzag chain propagating in the [001] direction. For 3b a combination of C—H...O and N—H...O intermolecular interactions link the molecules into a zigzag ribbon propagating in the [001] direction.
Crystal structure of 2-[(E)-2-(4-bromophenyl)diazen-1-yl]-4,5-bis(4-methoxyphenyl)-1H-imidazole: the first example of a structurally characterized triarylazoimidazole
Authors: Temesgen Ayalew , Tskhovrebov Alexander G. , Vologzhanina Anna V. , Le Tuan A. , Khrustalev Victor N.
Published: February 2021

The title compound, C23H19BrN4O2, is a product of an azo coupling reaction between 3,4-bis(4-methoxyphenyl)imidazole and 4-bromophenyldiazonium tetrafluoroborate. Its crystal structure was determined using data collected at 120 K. The molecule adopts a trans configuration with respect to the N=N double bond. The imidazole and aryl rings attached to the azo linkage are coplanar within 12.73 (14)°, which indicates significant electron delocalization within the molecule. In the crystal, the molecules form centrosymmetric dimers via pairs of N—H...O hydrogen bonds.
Crystal structure and Hirshfeld surface analysis of poly[[bis[μ4-N,N′-(1,3,5-oxadiazinane-3,5-diyl)bis(carbamoylmethanoato)]nickel(II)tetrapotassium] 4.8-hydrate]
Authors: Plutenko Maksym O. , Haukka Matti , Husak Alina O. , Iskenderov Turganbay S. , Mulloev Nurullo U.
Published: February 2021

The title compound, {[K4Ni2(C7H6N4O7)2]·4.8H2O}
n
, was obtained as a result of a template reaction between oxalohydrazidehydroxamic acid, formaldehyde and nickel(II) nitrate followed by partial hydrolysis of the formed intermediate. The two independent [Ni(C7H6N4O7)]2– complex anions exhibit pseudo-C
S symmetry and consist of an almost planar metal-containing fragment and a 1,3,5-oxadiazinane ring with a chair conformation disposed nearly perpendicularly with respect to the former. The central NiII atom has a square-planar N2O2 coordination arrangement formed by two amide N and two carboxylate O atoms. In the crystal, the nickel(II) complex anions form layers parallel to the ab plane. Neighboring complex anion layers are connected by layers of potassium cations for which two of the four independent cations are disordered over two sites [ratios of 0.54 (3):0.46 (3) and 0.9643 (15):0.0357 (15)]. The framework is stabilized by an extensive system of hydrogen bonds where the water molecules act as donors and the carboxylic O atoms, the amide O atoms and the oxadiazinane N atoms act as acceptors.
Synthesis and crystal structure of catena-poly[[tetra-μ-acetato-copper(II)]-μ-6-ethoxy-N
2,N
4-bis[2-(pyridin-2-yl)ethyl]-1,3,5-triazine-2,4-diamine]
Authors: Ayodele Mayokun J. , Green Travis C. , Warsapperuma W. A. Chathuri V. , Forbes Malcolm D. E. , Ostrowski Alexis D.
Published: February 2021

The title compound, [Cu2(C19H23N7O)(C2H3O2)4]
n
, was obtained via reaction of copper(II) acetate with the coordinating ligand, 6-ethoxy-N
2,N
4-bis[2-(pyridin-2-yl)ethyl]-1,3,5-triazine-2,4-diamine. The crystallized product adopts the monoclinic P21/c space group. The metal core exhibits a paddle-wheel structure typical for dicopper tetraacetate units, with triazine and pyridyl nitrogen atoms from different ligands coordinating to the two axial positions of the paddle wheel in an asymmetric manner. This forms a coordination polymer with the segments of the polymer created by the c-glide of the P21/c setting of the space group. The resulting chains running along the c-axis direction are held together by intramolecular N—H...O hydrogen bonding. These chains are further packed by dispersion forces, producing an extended three-dimensional structure.
1-{3-[(7-Fluoro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino]piperidin-1-yl}propan-1-one
Authors: Andreev Stanislav , Schollmeyer Dieter , Koch Pierre
Published: February 2021

The title compound, C19H22FN5O, has been synthesized as an inhibitor of glycogen synthase kinase-3β. Two molecules interact via two N—H...N hydrogen bonds, forming centrosymmetric dimers.
13-Benzyl-4,11-dihydroxy-1,8-diphenyl-2,9-dithia-13-azadispiro[4.1.4.3]tetradecan-6-one
Authors: Vinotha G. , Sundar T. V. , Sharmila N.
Published: February 2021

In the title compound, C30H31NO3S2, the piperidine ring adopts a distorted chair conformation. The thiophene rings have twisted conformations about the C—C bonds. The mean plane of the piperidine ring makes a near orthogonal conformation with the toluene ring. Two of the phenyl rings in the structure are positionally disordered over two sets of sites with occupancies of 0.56 (2)/0.44 (2) and 0.672 (16)/0.328 (16). A region of disordered electron density was corrected for using the SQUEEZE [Spek (2015). Acta Cryst. C71, 9–18] routine in PLATON. The given chemical formula and other crystal data do not take into account the unknown solvent molecule. In the crystal, O—H...O hydrogen bonds are observed along with intramolecular S...H, O...H, C...H and H...H contacts.