bluuebunny/crossref_metadata_2025 huggingface dataset:
Intra-dorsal striatal acetylcholine M1 but not dopaminergic D1 or glutamatergic NMDA receptor antagonists inhibit the consolidation of duration memory in interval timing
Authors: Nishioka Masahiko , Kamada Taisuke , Nakata Atsushi , Shiokawa Naoko , Kinoshita Aoi , Hata Toshimichi
Published: May 2021

The striatal beat frequency (SBF) model assumes that striatal medium spiny neurons encode duration via synaptic plasticity. Muscarinic 1 (M1) cholinergic receptors, as well as dopamine and glutamate receptors, are important for neural plasticity in the dorsal striatum. Therefore, we investigated the effect of inhibiting these receptors on the formation of duration memory. After sufficient training in a Peak interval (PI)-20 s procedure, rats were given a single or mixed infusion of a selective antagonist for the dopamine D1 receptor (SCH23390, 0.5 μg per side), the NMDA-type glutamate receptor (D-AP5, 3 μg), or the M1 receptor (pirenzepine, 10 μg) bilaterally in the dorsal striatum, immediately before starting a PI 40 s session (shift session). On the next day, the rats were tested for new duration memory (40 s) in a session in which no lever presses were reinforced (probe session). In the shift session, performance was tie, irrespective of the drug injected. However, in the probe session, the mean peak time (an index of duration memory) of the M1 + NMDA co-blockade group, but not of the D1 + NMDA co-blockade group, was lower than that of the control group (Exp. 1 and 2). In Exp. 3, the effect of the co-blockade of M1 and NMDA receptors was replicated. Moreover, sole blockade of M1 receptors induced the same effect as M1 and NMDA blockade. These results suggest that in the dorsal striatum, the M1 receptor, but not the D1 or NMDA receptors, are involved in the consolidation of duration memory.
Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury
Authors: Alhajlah Sharif , Thompson Adam M. , Ahmed Zubair
Published: May 2021

CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-66. Here, we focused on RTN3 as its contribution in CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury.
Isolation of SARS-CoV-2 B.1.1.28.2 P2 variant and pathogenicity comparison with D614G variant in hamster model
Authors: Yadav Pragya , Mohandas Sreelekshmy , Sarkale Prasad , Nyayanit Dimpal , Shete Anita , Sahay Rima , Potdar Varsha , Baradkar Shreekant , Gupta Nivedita , Sapkal Gajanan , Abraham Priya , Panda Samiran , Bhargava Balram
Published: May 2021

Background

Considering the potential threat from emerging SARS-CoV-2 variants and the rising COVID-19 cases, SARS-CoV-2 genomic surveillance is ongoing in India. We report herewith the isolation of the P.2 variant (B.1.1.28.2) from international travelers and further its pathogenicity evaluation and comparison with D614G variant (B.1) in hamster model.

Methods

Virus isolation was performed in Vero CCL81 cells and genomic characterization by next generation sequencing. The pathogenicity of the isolate was assessed in Syrian hamster model and compared with B.1 variant.

Results

B.1.1.28.2 variant was isolated from nasal/throat swabs of international travelers returned to India from United Kingdom and Brazil. The B.1.1.28.2 variant induced body weight loss, viral replication in the respiratory tract, lung lesions and caused severe lung pathology in infected Syrian hamster model in comparison, with B.1 variant infected hamsters. The sera from B.1.1.28.2 infected hamsters efficiently neutralized the D614G variant virus whereas 6-fold reduction in the neutralization was seen in case of D614G variant infected hamsters’ sera with the B.1.1.28.2 variant.

Conclusions

B.1.1.28.2 lineage variant could be successfully isolated and characterization could be performed. Pathogenicity of the isolate was demonstrated in Syrian hamster model and in comparison, with B.1 variant was found more pathogenic. The findings of increased disease severity and neutralization reduction is of great concern and point towards the need for screening the vaccines for efficacy.
Relationships between endogenous circadian period, physiological and cognitive parameters and sex in aged non-human primates
Authors: Hozer Clara , Pifferi Fabien
Published: May 2021

The biological clock exhibits circadian rhythms, with an endogenous period tau close to 24h. The circadian resonance theory proposes that lifespan is reduced when endogenous period goes far from 24h. It has been suggested that daily resetting of the circadian clock to the 24h external photoperiod might induce marginal costs that would accumulate over time and forward accelerate aging and affect fitness. In this study, we aimed to evaluate the link between the endogenous period and biomarkers of aging in order to investigate the mechanisms of the circadian resonance theory. We studied 39 middle-aged and aged Microcebus murinus, a nocturnal non-human primate whose endogenous period is about 23.1h, measuring the endogenous period of locomotor activity, as well as several physiological and behavioral parameters (rhythm fragmentation and amplitude, energetic expenditure, oxidative stress, insulin-like growth factor-1 (IGF-1) concentrations and cognitive performances) in both males and females. We found that aged males with tau far from 24h displayed increased oxidative stress. We also demonstrated a positive correlation between tau and IGF-1 concentrations, as well as learning performances, in males and females. Together these results suggest that a great deviation of tau from 24h leads to increased biomarkers of age-related impairments.

Summary Statement

This manuscript describes how the misalignment between the endogenous circadian period and the 24h of the environmental periodicity impacts aging biomarkers and accelerates cognitive decline.
Comprehensive Analysis of Spatial Architecture in Primary Liver Cancer
Authors: Wu Rui , Guo Wenbo , Qiu Xinyao , Wang Shicheng , Sui Chengjun , Lian Qiuyu , Wu Jianmin , Shan Yiran , Yang Zhao , Yang Shuai , Wu Tong , Wang Kaiting , Zhu Yanjing , Wang Shan , Liu Changyi , Zhang Yangqianwen , Zheng Bo , Li Zhixuan , Zhang Yani , Shen Siyun , Zhao Yan , Wang Wenwen , Bao Jinxia , Hu Ji , Wu Xuan , Jiang Xiaoqing , Wang Hongyang , Gu Jin , Chen Lei
Published: May 2021

Heterogeneity is the major challenge for cancer prevention and therapy. Here, we firstly constructed high-resolution spatial transcriptomes of primary liver cancers (PLCs) containing 84,823 spots within 21 tissues from 7 patients. The sequential comparison of spatial tumor microenvironment (TME) characteristics from non-tumor to leading-edge to tumor regions revealed that the tumor capsule potentially affects intratumor spatial cluster continuity, transcriptome diversity and immune cell infiltration. Locally, we found that the bidirectional ligand-receptor interactions at the 100 μm wide cluster-cluster boundary contribute to maintaining intratumor architecture. Our study provides novel insights for diverse tumor ecosystem of PLCs and has potential benefits for cancer intervention.
The Molecular Logic Organizing the Functional Compartmentalization of Reciprocal Synapses
Authors: Wang Cosmos Yuqi , Trotter Justin H. , Liakath-Ali Kif , Lee Sung-Jin , Liu Xinran , Südhof Thomas C.
Published: May 2021

Reciprocal synapses are formed by neighboring dendritic processes that create the smallest possible neural circuit. Reciprocal synapses are widespread in brain and essential for information processing, but constitute a conceptual conundrum: How are adjacent pre- and post-synaptic specializations maintained as separate functional units? Here, we reveal an organizational principle for reciprocal synapses, using dendrodendritic synapses between mitral and granule cells in the mouse olfactory bulb as a paradigm. We show that mitral cells secrete cerebellin-1 to block thecis-interaction of mitral cell neurexins with neuroligins, thereby enabling their separatetrans-interactions. Ablating either cerebellin-1 or neuroligins in mitral cells severely impaired granule cell→mitral cell synapses, as did overexpression of postsynaptic neurexins that formcis-complexes with neuroligins, but not of mutant neurexins unable to bind to neuroligins. Our data uncover acis/trans-protein interaction network as a general design principle that organizes reciprocal dendro-dendritic synapses by compartmentalizing neurexin-basedtrans-synaptic protein complexes.
CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation
Authors: Tooley John G. , Catlin James P. , Schaner Tooley Christine E.
Published: May 2021

The N-terminal methyltransferase NRMT1 is an important regulator of protein-DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked Pax7 expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.
A rapid and reliable multiplexed LC-MS/MS method for simultaneous analysis of six monoamines from brain tissues
Authors: Davla Sejal , Daly Edward , Nedow Jenn , Gritsas Ari , Curran Laura , Taylor Lorne , van Meyel Donald J.
Published: May 2021

Monoamines are a class of neuromodulators that are crucial for a variety of brain functions, including control of mood, movement, sleep and cognition. From mammals to insects, the nervous system is enriched in monoamines such as dopamine, serotonin and melatonin, analytes which range from being highly polar to non-polar. Here we developed a method using liquid chromatography coupled with mass spectrometry (LC-MS) to quantify in a single run the amounts of six distinct monoamines in extracts from dissected Drosophila and mouse brain tissues. The measured monoamines were dopamine (DA), serotonin (also known as 5-hydroxytryptamine (5-HT)), octopamine (OA, an insect equivalent of norepinephrine), tyramine (TA), melatonin (MT) and N-acetyl-hydroxy-serotonin (NAS). The analytical range of these monoamines was between 0.25 to 5.0 ng/mL.
HIV-1 Nef and CycK:CDK13 antagonize SERINC5 for optimal viral infectivity
Authors: Chai Qingqing , Li Sunan , Collins Morgan K. , Li Rongrong , Ahmad Iqbal , Johnson Silas F. , Frabutt Dylan A. , Yang Zhichang , Shen Xiaojing , Sun Liangliang , Hu Jian , Hultquist Judd F. , Matija Peterlin B. , Zheng Yong-Hui
Published: May 2021

HIV-1 Nef antagonizes SERINC5 by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identified cyclin K and cyclin-dependent kinase 13 (CycK:CDK13) as a new Nef-associated kinase complex. CycK:CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counter activity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we created chimeric proteins between CD8 and SERINC5. Nef not only downregulates, but importantly, also binds to this chimera in a S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.
Cancer Prognosis According to Parthanatos Features
Authors: Messikommer Alessandra , Maru Bruktawit , Seipel Katja , Valk Peter J. M. , Theocharides Alexandre P.A. , Pabst Thomas , McKeague Maureen , Luedtke Nathan W.
Published: May 2021

For nearly 50 years, translational research studies aimed at improving chemotherapy-induced killing of cancer cells have focused on the induction of apoptosis. Here we show that a PARP-1-mediated programmed cell death mechanism “parthanatos” is associated with the successful, front-line treatment of a common cancer. Peripheral blood mononuclear cells (PBMCs) from healthy human donors (10 of 10 tested), as well as primary cancer cells from approximately 50% of acute myeloid leukemia (AML) patients (n = 18 of 39 tested, French-American-British (FAB) subtypes M4 and M5) exhibited two distinctive features of parthanatos upon treatment with a front-line drug combination of cytarabine and an anthracycline. Statistically significant improvements in survival rates were observed in the parthanatos positive versus parthanatos negative AML patient groups (HR = 0.22 – 0.38, p = 0.002 – 0.05). Near-median expression of PARP1 mRNA was associated with a 50% longer survival time (HR = 0.66, p = 0.01), and the poly [ADP-ribose] polymerase (PARP) inhibitor Olaparib exhibited antagonistic activities against ara-C and idarubicin in primary blood monocytes from healthy donors as well as primary cancer isolates from ~50% of AML patients. Together these results suggest that PARP activity is a prognostic biomarker for AML subtypes M4 and M5 and support the relevance of parthanatos in curative chemotherapy of AML.

In Brief

Messikommer and co-workers report that PARP-1-mediated programmed cell death is associated with successful, front-line treatment of acute myeloid leukemia (AML).

Highlights

The first-line cancer drug cytarabine (ara-C) induces parthanatos or apoptosis, depending on the specific AML cell line being treated.

OCI-AML3 cells undergo parthanatos or apoptosis, depending on the specific drug being added.

The presence of two parthanatos features in primary cancer cells from AML patients (n = 18 of 39 tested) having French-American-British (FAB) subclassifications M4 or M5 is associated with four-fold improved survival (HR = 0.23, p = 0.01) following curative chemotherapy with ara-C and an anthracycline.

The poly [ADP-ribose] polymerase (PARP) inhibitor Olaparib exhibits antagonistic activities against ara-C and idarubicin in primary blood monocytes from healthy donors as well as primary cancer isolates from ~50% of AML patients.

Near-median expression of PARP1 mRNA is associated with a 50% increase in survival time (HR = 0.66, p = 0.01) of AML patients following chemotherapy with ara-C and idarubicin.

GRAPHICAL ABSTRACT
The RNA chaperone protein CspA stimulates translation during cold acclimation by promoting the progression of the ribosomes
Authors: Giuliodori Anna Maria , Belardinelli Riccardo , Duval Melodie , Garofalo Raffaella , Schenckbecher Emma , Hauryliuk Vasili , Ennifar Eric , Marzi Stefano
Published: May 2021

CspA is an RNA binding protein expressed during cold-shock in Escherichia coli, capable of stimulating translation of several mRNAs – including its own – at low temperature. We used reconstituted translation systems to monitor the effects of CspA on the different steps of the translation process and probing experiments to analyze the interactions with its target mRNAs. We specifically focused on cspA mRNA which adopts a cold-induced secondary structure at temperatures below 20°C and a more closed conformation at 37°C. We show that at low temperature CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome (37°C form). CspA interacts with its mRNA without inducing large structural rearrangement, does not bind the ribosomal subunits and is not able to stimulate the formation of the translation initiation complexes. On the other hand, CspA promotes the progression of the ribosomes during translation of its mRNA at low temperature and this stimulation is mRNA structure-dependent. A similar structure-dependent mechanism may be responsible for the CspA- dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.
Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy
Authors: Nino Daniel F. , Milstein Joshua N.
Published: May 2021

In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks, and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on dSTORM datasets of photo-switching Alexa647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.
The molecular mechanism of load adaptation by branched actin networks
Authors: Li Tai-De , Bieling Peter , Weichsel Julian , Mullins R. Dyche , Fletcher Daniel A.
Published: May 2021

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes. Load forces slow the growth and increase the density of these networks, but the molecular mechanisms governing this force response are not well understood. Here we use single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Unexpectedly, force slows the rate of filament nucleation by promoting the interaction of nucleation promoting factors with actin filament ends, limiting branch formation. This inhibition is countered by an even larger force-induced drop in the rate of filament capping, resulting in a shift in the balance between nucleation and capping that increases network density. Remarkably, the force dependence of capping is identical to that of filament elongation because they require the same size gap to appear between the filament and load for insertion. These results provide direct evidence that Brownian Ratchets generate force and govern the load adaptation of branched actin networks.
A Neural Model of RNA Splicing: Learning Motif Distances with Self-Attention and Toeplitz Max Pooling
Authors: Quinn Thomas P. , Nguyen Dang , Gupta Sunil , Venkatesh Svetha
Published: May 2021

Alternative RNA splicing is an important regulator of tissue development and specificity, and a relevant mechanism in cancer progression. There exists a strong motivation to disentangle the rules that govern RNA splicing, in part because this knowledge may one day yield new clinically relevant diagnostic tools and therapeutics. It is no easy to task to reverse engineer how the splicesome machinery choreographs the removal and addition of RNA elements following transcription. Here, we propose an interpretable neural network called the Toeplitz ATtention Architecture (TATA), which learns distance-dependent motif interactions through a novel Toeplitz max pool layer that captures the relative distance between interacting CNN filters. TATA is a completely transparent “clear-box” solution: every model parameter is human-interpretable. We validate TATA on simulated data, then apply it to real data to identify putative cis-regulatory elements that interact with primary RNA splice sites.
Structural basis for DNA targeting by the Tn7 transposon
Authors: Shen Yao , Gomez-Blanco Josue , Petassi Michael T. , Peters Joseph E. , Ortega Joaquin , Guarné Alba
Published: May 2021

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7-family utilize a AAA+ adaptor (TnsC) to coordinates target-site selection with transposase activation and prevent insertions at sites already containing a Tn7 element. Due to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. We find that TnsC forms an asymmetric ring on target DNA that segregates target-site selection and transposase recruitment to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP-hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.
Disc Large Homolog 1 is critical for early microcluster formation and activation in human T cells
Authors: June Guha , Raj Chari
Published: May 2021

T cell activation by antigen involves multiple sequential steps, including TCR-microcluster (MC) formation, immunological synapse formation and phosphorylation of mediators downstream of the TCR. The adaptor protein Disc Large Homolog 1 (DLG1) is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. Here, we report using knockdown and knockout studies in human T cells that DLG1 functions even earlier to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-ζ chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-ζ. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells.
Phase variable expression of pdcB, a phosphodiesterase influences sporulation in Clostridioides difficile
Authors: Dhungel Babita Adhikari , Govind Revathi
Published: May 2021

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to hypervirulence strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovered that PdcB, a phosphodiesterase to influence sporulation in C. difficile UK1 strain positively. Through genetic and biochemical assays, we have shown that phase variable expression of pdcB results in hypo- and hyper-sporulation phenotype. In the “ON” orientation, the identified promotor is the right orientation to drive the expression of pdcB. Production of PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP concentration, resulting in hyper-sporulation phenotype. The OFF orientation of pdcB switch or mutating pdcB results in increased cyclic-di-GMP and hypo-sporulating phenotype. Additionally, we demonstrated that CodY binds to the upstream region of pdcB to represses its expression, and CodY mediated repression is relieved by the DNA inversion.
Statistical Analysis of Three-Dimensional Chromatin Packing Domains Determined by Chromatin Scanning Transmission Electron Microscopy (ChromSTEM)
Authors: Li Yue , Agrawal Vasundhara , Virk Ranya K. A. , Roth Eric , Li Wing Shun , Eshein Adam , Frederick Jane , Huang Kai , Almassalha Luay , Bleher Reiner , Carignano Marcelo A. , Szleifer Igal , Dravid Vinayak P. , Backman Vadim
Published: May 2021

Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.
An hidden Markov model to estimate homozygous-by-descent probabilities associated with nested layers of ancestors
Authors: Druet Tom , Gautier Mathieu
Published: May 2021

Inbreeding results from the mating of related individuals and has negative consequences because it brings together deleterious variants in one individual. Genomic estimates of the inbreeding coefficients are preferred to pedigree-based estimators as they measure the realized inbreeding levels and they are more robust to pedigree errors. Several methods identifying homozygous-by-descent (HBD) segments with hidden Markov models (HMM) have been recently developed and are particularly valuable when the information is degraded or heterogeneous (e.g., low-fold sequencing, low marker density, heterogeneous genotype quality or variable marker spacing). We previously developed a multiple HBD class HMM where HBD segments are classified in different groups based on their length (e.g., recent versus old HBD segments) but we recently observed that for high inbreeding levels with many HBD segments, the estimated contributions might be biased towards more recent classes (i.e., associated with large HBD segments) although the overall estimated level of inbreeding remained unbiased. We herein propose a new model in which the HBD classification is modeled in successive nested levels with decreasing expected HBD segment lengths, the underlying exponential rates being directly related to the number of generations to the common ancestor. The non-HBD classes are now modeled as a mixture of HBD segments from later generations and shorter non-HBD segments (i.e., both with higher rates). The new model has improved statistical properties and performs better on simulated data compared to our previous version. We also show that the parameters of the model are easier to interpret and that the model is more robust to the choice of the number of classes. Overall, the new model results in an improved partitioning of inbreeding in different HBD classes and should be preferred.
Optimized liquid and gas phase fractionation increase HLA-peptidome coverage for primary cell and tissue samples
Authors: Klaeger Susan , Apffel Annie , Clauser Karl R. , Sarkizova Siranush , Oliveira Giacomo , Rachimi Suzanna , Le Phuong M. , Tarren Anna , Chea Vipheaviny , Abelin Jennifer G. , Braun David A. , Ott Patrick A. , Keshishian Hasmik , Hacohen Nir , Keskin Derin B. , Wu Catherine J. , Carr Steven A.
Published: May 2021

Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require many millions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens.
Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors
Authors: Carter Angela M. , Kumar Nilesh , Herring Brendon , Tan Chunfeng , Guenter Racheal , Telange Rahul , Howse Wayne , Viol Fabrice , Graham Chris , McCaw Tyler R. , Bickerton Hayden , Gillardon Frank , Woltering Eugene A. , Dhall Deepti , Totenhagen John , Banerjee Ronadip , Kurian Elizabeth , Reddy Sushanth , Chen Herbert , Schrader Joerg , Rose J. Bart , Mukhtar M. Shahid , Bibb James A.
Published: May 2021

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bitransgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in β–islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. The utility of this model is enhanced by ability to form tumor-derived allografts. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.
Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data
Authors: Dimchev Georgi , Amiri Behnam , Fäßler Florian , Falcke Martin , Schur Florian KM
Published: May 2021

A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. However, this also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of filamentous networks from cryo-ET datasets to facilitate the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the manipulation of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner.
Machine learning approaches to predict the plant-associated phenotype of Xanthomonas strains
Authors: Molder Dennie te , Poncheewin Wasin , Schaap Peter J. , Koehorst Jasper J.
Published: May 2021

The genus Xanthomonas has long been considered to consist predominantly of plant pathogens, but over the last decade there has been an increasing number of reports on non-pathogenic and endophytic members. As Xanthomonas species are prevalent pathogens on a wide variety of important crops around the world, there is a need to distinguish between these plant-associated phenotypes. To date a large number of Xanthomonas genomes have been sequenced, which enables the application of machine learning (ML) approaches on the genome content to predict this phenotype. Until now such approaches to the pathogenomics of Xanthomonas strains have been hampered by the fragmentation of information regarding strain pathogenicity over many studies. Unification of this information into a single resource was therefore considered to be an essential step. Mining of 39 papers considering both plant-associated phenotypes, allowed for a phenotypic classification of 578 Xanthomonas strains. For 65 plant-pathogenic and 53 non-pathogenic strains the corresponding genomes were available and de novo annotated for the presence of Pfam protein domains used as features to train and compare three ML classification algorithms; CART, Lasso and Random Forest. Recursive feature extraction provided further insights into the virulence enabling factors, but also yielded domains linked to traits not present in pathogenic strains.
Phototropin-mediated perception of light direction in Arabidopsis leaves regulates blade flattening
Authors: Legris Martina , Szarzynska-Erden Bogna Maria , Trevisan Martine , Allenbach-Petrolati Laure , Fankhauser Christian
Published: May 2021

One conserved feature among angiosperms is the development of flat thin leaves. This developmental pattern optimizes light capture and gas exchange for photosynthesis. The blue light receptors phototropins are required for leaf flattening, with the null phot1phot2 mutant showing downwards curled leaves in Arabidopsis. However, key aspects of their function in leaf development remain unknown. Here, we performed a detailed spatiotemporal characterization of phototropin function in Arabidopsis leaves. We found that phototropins perceive light direction in the leaf blade, and similar to their role in hypocotyls they control the spatial pattern of auxin signaling possibly modulating auxin transport, to ultimately regulate cell expansion. Phototropin signaling components in the leaf partially differ from hypocotyls. Moreover, the light response on the upper (adaxial) and lower (abaxial) sides of the leaf blade suggest a partially distinct requirement of phototropin signaling components on each side. In particular, NON PHOTOTROPIC HYPOCOTYL 3 (NPH3) showed an adaxial-specific function. In addition, we show a prominent role of PHYTOCHROME KINASE SUBSTRATE 3 (PKS3) in leaf flattening. Among the auxin transporters tested, PINs and AUX/LAX influence the response most prominently. Overall, our results show that directional blue light perception by the phototropins is a key aspect of leaf development, integrating endogenous and exogenous signals.

One sentence summary

Phototropins perceive light direction in the leaf and control the auxin signaling pattern to regulate blade flattening.
Characterizing spatial gene expression heterogeneity in spatially resolved single-cell transcriptomic data with nonuniform cellular densities
Authors: Miller Brendan F. , Bambah-Mukku Dhananjay , Dulac Catherine , Zhuang Xiaowei , Fan Jean
Published: May 2021

Recent technological advances have enabled spatially resolved measurements of expression profiles for hundreds to thousands of genes in fixed tissues at single-cell resolution. However, scalable computational analysis methods able to take into consideration the inherent 3D spatial organization of cell types and nonuniform cellular densities within tissues are still lacking. To address this, we developed MERINGUE, a computational framework based on spatial autocorrelation and cross-correlation analysis to identify genes with spatially heterogeneous expression patterns, infer putative cell–cell communication, and perform spatially informed cell clustering in 2D and 3D in a density-agnostic manner using spatially resolved transcriptomic data. We applied MERINGUE to a variety of spatially resolved transcriptomic data sets including multiplexed error-robust fluorescence in situ hybridization (MERFISH), spatial transcriptomics, Slide-seq, and aligned in situ hybridization (ISH) data. We anticipate that such statistical analysis of spatially resolved transcriptomic data will facilitate our understanding of the interplay between cell state and spatial organization in tissue development and disease.
Design and use of a sapphire single-crystal gas-pressure cell forin situneutron powder diffraction
Authors: Finger Raphael , Kurtzemann Nadine , Hansen Thomas C. , Kohlmann Holger
Published: May 2021

A sapphire single-crystal gas-pressure cell without external support allowing unobstructed optical access by neutrons has been developed and optimized for elasticin situneutron powder diffraction using hydrogen (deuterium) gas at the high-intensity two-axis diffractometer D20 at the Institut Laue-Langevin (Grenoble, France). Given a proper orientation of the single-crystal sample holder with respect to the detector, parasitic reflections from the sample holder can be avoided and the background can be kept low. Hydrogen (deuterium) gas pressures of up to 16.0 MPa at 298 K and 8.0 MPa at 655 K were tested successfully for a wall thickness of 3 mm. Heating was achieved by a two-sided laser heating system. The typical time resolution ofin situinvestigations of the reaction pathway of hydrogen (deuterium) uptake or release is on the order of 1 min. Detailed descriptions of all parts of the sapphire single-crystal gas-pressure cell are given, including materials information, technical drawings and instructions for use.
Relativistic Hirshfeld atom refinement of an organo-gold(I) compound
Authors: Pawlędzio Sylwia , Malinska Maura , Woińska Magdalena , Wojciechowski Jakub , Andrade Malaspina Lorraine , Kleemiss Florian , Grabowsky Simon , Woźniak Krzysztof
Published: May 2021

The main goal of this study is the validation of relativistic Hirshfeld atom refinement (HAR) as implemented in Tonto for high-resolution X-ray diffraction datasets of an organo-gold(I) compound. The influence of the relativistic effects on statistical parameters, geometries and electron density properties was analyzed and compared with the influence of electron correlation and anharmonic atomic motions. Recent work in this field has indicated the importance of relativistic effects in the static electron density distribution of organo-mercury compounds. This study confirms that differences in electron density due to relativistic effects are also of significant magnitude for organo-gold compounds. Relativistic effects dominate not only the core region of the gold atom, but also influence the electron density in the valence and bonding region, which has measurable consequences for the HAR refinement model parameters. To study the effects of anharmonic motion on the electron density distribution, dynamic electron density difference maps were constructed. Unlike relativistic and electron correlation effects, the effects of anharmonic nuclear motion are mostly observed in the core area of the gold atom.
Structural reasons for the formation of multicomponent products and the influence of high pressure
Authors: Konieczny Krzysztof A. , Bąkowicz Julia , Paliwoda Damian , Warren Mark R. , Ciesielski Arkadiusz , Cyrański Michał K. , Turowska-Tyrk Ilona
Published: May 2021

(S)-(−)-1-Phenylethanaminium 4-(2,4,6-triisopropylbenzoyl)benzoate (S-PEATPBB) undergoes a photochemical reaction in its crystalline form upon UV irradiation and forms three different products: the first product is the result of a Yang cyclization with the participation of the δ-H atom of o-isopropyl (product D) and the second and third products are obtained via a Norrish–Yang reaction with the involvement of the γ-H atom of 2-isopropyl (product P) and 6-isopropyl (product Z). These products are formed in different proportions (D > P >> Z). The path and kinetics of the reaction were monitored step-by-step using crystallographic methods, both under ambient and high-pressure conditions. The reactivity of S-PEATPBB depends strongly on the geometry of the reaction centre and the volume of the reaction cavity. Due to the geometrical preferences making the cyclization reaction easier to proceed, product D dominates over the other products, while the formation of product Z becomes difficult or almost impossible at high pressure. The reaction proceeds with an increase of the unit-cell volume, which, suppressed by high pressure, results in a significant decrease of the reaction rate. The crystal lattice of S-PEATPBB shows high elasticity. The quality of the partially reacted crystal remains the same after decompression from 0.75 GPa to 0.1 MPa.
Entrepreneurial intention among Latin American university students
Authors: Leiva Juan Carlos , Mora-Esquivel Ronald , Krauss-Delorme Catherine , Bonomo-Odizzio Adriana , Solís-Salazar Martín
Published: May 2021

Purpose

This paper analyses how contextual factors at universities (entrepreneurship education and program learning) and cognitive variables (perceived behavioral control, implementation intentions, and attitude) influence entrepreneurial intentions among Latin American university students.

Design/Methodology/Approach

The empirical analysis employs a multilevel (hierarchical) linear model with a sample size of 9012 university students taken in 2018 from nine Latin American countries: Argentina, Brazil, Chile, Colombia, Costa Rica, Ecuador, México, Panamá, and Uruguay.

Findings

Overall, the university context and cognitive variables contribute to explaining entrepreneurial intentions in university students. Whereas program learning constitutes a variable that directly and indirectly explains entrepreneurial intentions among university students, attending entrepreneurship courses negatively influences their entrepreneurial intentions.

Originality/value

A central premise of this study is that the entrepreneurial process in university students is a multilevel phenomenon, given that university context and cognitive variables are key factors in entrepreneurial intentions. The findings support this premise and contribute to the existing literature on entrepreneurship in emerging economies. Nevertheless, the results reveal a more nuanced picture regarding the role of university context on the entrepreneurial intentions of students.
Social dimensions of synthetic biology in the agrifood sector: the perspective of Chinese and EU scientists
Authors: Jin Shan , Clark Beth , Li Wenjing , Kuznesof Sharron , Frewer Lynn J.
Published: May 2021

Purpose

Scientists' perceptions of societal needs and priorities will shape the innovation trajectories of synthetic biology (SB). In turn, these will be shaped by the funding and regulatory environments in which their research is conducted. This study intends to investigate scientists' perspectives on co-innovation with the public regarding implementation of pathways associated with SB including its agrifood applications.

Design/methodology/approach

Semi-structured interviews were conducted with Chinese and EU scientists (N = 9 and 13, respectively). Six prominent themes emerged from the data based on thematic analysis method.

Findings

Both Chinese and EU scientists regarded SB as being high-benefit, low-risk and ethically acceptable, and predicted its rejection by the general public and attributed this to the public's knowledge deficit and irrationality. They endorsed the deficit model of science communication, independent of greater emphasis on responsible research and innovation (RRI) in EU research projects. The findings raised concerns that public fears might intensify once they have learned about scientists' biased risk perceptions of SB; this calls for better involvement of broader stakeholders.

Research limitations/implications

As the sample size is relatively small, the generalisation of research findings needs to be cautious. However, the authors believe the findings have provided some insights that support increasingly RRI associated with SB.

Originality/value

This study has presented scientists' misunderstandings of societal responses to SB and science communication. It has also provided information to understand how SB-related issues and agenda can be better shaped in future.