bluuebunny/crossref_metadata_2025 huggingface dataset:
Trends and Relationships in Opioid Prescribing Rates and Overdose Rates in the United States 2013-2019
Authors: Enthoven Luke F.
Published: December 2022

The opioid epidemic began more than two decades ago and continues to increase in severity with the rate of overdose death increasing steadily. The epidemic was born in problematic claims about opioids and aggressive prescribing. After initial analysis of the first decade of the epidemic it became clear that actions would need to be taken to curtail overprescribing and many states passed legislation tracking and limiting opioid prescribing. Since then opioid prescribing has declined, but overdose has continued to increase at an alarming rate. This analysis is focused on evaluating more recent trends in opioid prescribing and overdose to see if the close positive correlation that existed between these values prior to 2010 holds true for the following decade. My findings indicate that between 2013 and 2019 that there is no relationship, or possibly a negative relationship, between opioid prescribing and overdose. Between 2013 and 2019 there was a 25% decrease in opioid prescribing rate yet a 96% increase in opioid overdose deaths. The close relationship that existed between prescribing and overdose appears to have diverged to the point where overdose increases as prescribing decreases. Linear regression on both national and state data revealed a significant negative relationship between overdose and prescribing for national data between 2013 and 2019 (R2value of 0.84 and a p-value of 0.003) and no relationship between overdose and prescribing for state data between 2014 and 2019 (R2value of 0.04 and a p-value of < 0.001). This questions the value of further efforts to reduce prescribing in preventing overdose and supports the hypothesis that restriction of prescription opioids can contribute to increased use of dangerous illicit opioids.
Nitrate Assimilation Underlying Kleptoplasty
Authors: Maruyama Moe , Kagamoto Tsuyoshi , Matsumoto Yuga , Onuma Ryo , Miyagishima Shin-ya , Tanifuji Goro , Nakazawa Masami , Kashiyama Yuichiro
Published: December 2022

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here we focused on the nitrogen metabolism ofRapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates,R. viridisexploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data ofR. viridis, we identified the gene RvNaRL, which had sequence similarity to nitrate reductases found in plants. Phylogenetic analysis revealed that RvNaRLwas acquired by a horizontal gene transfer event. To verify its function of the protein productRvNaRL, we established a RNAi mediated knockdown and a CRISPR-Cas9-mediated knockout experiments for the first time inR. viridisand applied them to this gene. The RvNaRLknockdown and knockout cells exhibited significant growth only when ammonium was supplied but, in contrast to the wild-type cells, no substantial growth when nitrate was supplied. Such arrested growth in absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains as observed. These results indicate thatRvNaRL is certainly involved in nitrate assimilation byR. viridis. Thus, we infer thatR. viridisachieved its advanced kleptoplastic strategy owing to a posteriori acquisition of the nitrate assimilation pathway the horizontal gene transfer.
Elucidation of Amyloid-Beta’s Gambit in Oligomerization: Truncated Aβ fragments of residues Aβ1-23, Aβ1-24 and Aβ1-25 rapidly seed to form SDS-stable, LMW Aβ oligomers that impair synaptic plasticity
Authors: Gil Beatriz , Rose Jamie , Demurtas Davide , Mancini Gian-Filippo , Sordet-Dessimoz Jessica , Sorrentino Vincenzo , Rudinskiy Nikita , Frosch Matthew P. , Hyman Bradley T. , Moniatte Marc , Spires-Jones Tara. L. , Herron Caroline E. , Schmid Adrien W.
Published: December 2022

In Alzheimer’s disease (AD), Amyloid-beta (Aβ) oligomers are considered an appealing therapeutic- and diagnostic target. However, to date, the molecular mechanisms associated with the pathological accumulation or structure of Aβ oligomers remains an enigma to the scientific community. Here we demonstrate the strong seeding properties of unique Aβ fragment signatures and show that the truncated Aβ peptides of residues Aβ1-23, Aβ1-24 and Aβ1-25, rapidly seed to form small, SDS-PAGE stable assemblies of ∼5kDa to ∼14kDa molecular mass range. Mass spectrometry analysis of SDS-PAGE fractionated and gel extracted oligomers revealed that the truncated Aβ isoforms of residues 1-23 to 1-25 form stable entities with low molecular weight (LMW) oligomers, which strongly resemble the regularly reported Aβ entities of putative dimeric or trimeric assemblies found in human post-mortem AD and Tg mouse brain extracts. Furthermore, electrophysiological recordings in the mouse hippocampus indicate that LMW Aβ assemblies formed by fragments Aβ1-23 to Aβ1-25 significantly impair long-term-potentiation (LTP) in the absence of full-length Aβ1-42. Extensive antibody screening highlights the important observation, that the LMW Aβ assemblies formed by these truncated Aβ peptides escape immuno-detection using conventional, conformation specific antibodies but, more importantly, the clinical antibody aducanumab. Our novel findings suggest that there are new Aβ target “loopholes” which can be exploited for the development of therapeutic antibodies with binding properties against stable target hotspots present in Aβ oligomers. We provide here a first example of a new class of monoclonal antibody with unique binding properties against LMW Aβ oligomers, in the absence of binding to large fibrillar Aβ assemblies, or dense amyloid plaques. Our research supports a novel, unparalleled approach for targeting early, pathological Aβ species during the insidious phase of AD and prior to the appearance of large oligomeric or protofibrilar assemblies.
A scoping review on the use of consumer-grade EEG devices for research
Authors: Sabio Joshua , Williams Nikolas S , McArthur Genevieve M , Badcock Nicholas A
Published: December 2022

BACKGROUND

Commercial electroencephalography (EEG) devices have become increasingly available over the last decade. These devices have been used in a wide variety of fields ranging from engineering to cognitive neuroscience.

PURPOSE

The aim of this study was to chart peer-review articles that used currently available consumer-grade EEG devices to collect neural data. We provide an overview of the research conducted with these relatively more affordable and user-friendly devices. We also inform future research by exploring the current and potential scope of consumer-grade EEG.

METHODS

We followed a five-stage methodological framework for a scoping review that included a systematic search using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) guidelines. We searched the following electronic databases: PsycINFO, MEDLINE, Embase, Web of Science, and IEEE Xplore. We charted study data according to application (BCI, experimental research, validation, signal processing, and clinical) and location of use as indexed by the first author’s country.

RESULTS

We identified 916 studies that used data recorded with consumer-grade EEG: 531 were reported in journal articles and 385 in conference papers. Emotiv devices were most used, followed by the NeuroSky MindWave, OpenBCI, interaXon Muse, and MyndPlay Mindband. The most common use was for brain-computer interfaces, followed by experimental research, signal processing, validation, and clinical purposes.

CONCLUSIONS

Consumer-grade EEG has proven to be a useful tool for neuroscientific research and will likely continue to be used well into the future. Our study provides a comprehensive review of their application, as well as future directions for researchers who wish to use these devices.
A highly activeBurkholderiapolyketoacyl-CoA thiolase for production of triacetic acid lactone
Authors: Wang Zilong , Cheong Seokjung , Pereira Jose Henrique , Kim Jinho , DeGiovanni Andy , Guo Yifan , Lan Guangxu , Barcelos Carolina Araujo , Haushalter Robert , Lee Taek Soon , Adams Paul D. , Keasling Jay D.
Published: December 2022

Triacetic acid lactone (TAL) is a platform chemical biosynthesized primarily through decarboxylative Claisen condensation by type III polyketide synthase 2-pyrone synthase (2-PS). However, this reaction suffers from intrinsic energy inefficiency and feedback inhibition by and competition for malonyl-CoA. TAL production through non-decarboxylative Claisen condensation by polyketoacyl-CoA thiolase alleviates many of these disadvantages. We discovered five more thiolases with TAL production activity by exploring homologs of a previously reported polyketoacyl-CoA thiolase, BktB, fromCupriavidus necator. Among them, the BktB homolog fromBurkholderiasp. RF2-non_BP3 has ∼ 30 times higherin vitroandin vivoTAL production activity and led to ∼10 times higher TAL titer than 2-PS when expressed inEscherichia coli, achieving a titer of 2.8 g/L in fed-batch fermentations. This discovery of a novel polyketoacyl-CoA thiolase with superior TAL production activity paves the way for realization of total biomanufacturing of TAL.
What are we learning with Yoga: a text mining approach to literature
Authors: Raittz Rosangela Ieger , De Pierri Camilla R. , Perico Camila P. , Machado Diogo J. , Marchaukoski Jeroniza N. , Raittz Roberto T.
Published: December 2022

Techniques used in yoga existed long before science as we know it. However, with Yoga westernization, the proliferation of scientific studies addressing the subject is exponential. Even though the literature presents thousands of related articles, many studies are considered flimsy — the diversity of methods, areas of interest, and focus cause it to become a complex environment without general guidelines for the researcher. This status may represent one of the impediments to the field’s evolution. This study aims to elaborate a global structure of the literature on Yoga to open the door for multidisciplinary collaboration and bring new light to some relevant aspects. Due to its complexity, we understand that techniques to model complex domains are necessary. Contemporary methods of Artificial Intelligence have promoted advances in Bioinformatics, including text mining to scientific literature. Through the vector representation of texts, we got semantic abilities and could organize data in a way that allowed us to acquire a global vision of this literature. Additionally, we made the platforms available to Yoga researchers to enable them to evaluate our findings and make their forays into the literature. Besides better understanding why we study Yoga, we discovered that the literature offers insights into the relationships in broader aspects.
Dissecting the cell of origin of aberrant SALL4 expression in myelodysplastic syndrome
Authors: Tatetsu Hiro , Watanabe Miho , Liu Jun , Tokunaga Kenji , Iwanaga Eisaku , Komohara Yoshihiro , Thrash Emily , Masao Matsuoka , Tenen Daniel G. , Chai Li
Published: December 2022

Myelodysplastic syndrome (MDS) is a group of heterogeneous diseases characterized by cytologic dysplasia and cytopenias resulting from ineffective hematopoiesis. Oncofetal protein SALL4 is a known oncogene in MDS and its baseline expression level serves as a prognostic biomarker for MDS at the time of diagnosis. In addition, a recent study showed that SALL4 upregulation following hypomethylating agent treatment in MDS patients correlates with poor outcomes. Despite its important mechanistic and diagnostic significance, the cellular identity of bone marrow cells with aberrant SALL4 expression in MDS patients remains unknown.

In this study, we analyzed MDS bone marrow cells on single cell level by mass cytometry (CyTOF) and found that SALL4 was mainly aberrantly expressed in the hematopoietic stem and progenitor cells (HSPC) as well as myeloid lineages. Within the HSPC population from MDS patients, SALL4 and p53 were co-expressed, with the highest co-expressing clones harboring pathogenic TP53 mutations. Overall, our study characterizes for the first time the aberrant SALL4 expression in primary MDS patient samples at a single-cell level. Further studies on the SALL4/p53 network for in-depth mechanistic investigation are needed in the future.

Key Points

SALL4 expression in various MDS BM cells confirmed by mass cytometry (CyTOF). SALL4 and p53 double positive cells were predominantly found in the hematopoietic stem and progenitor cell (HSPC) population and associated with pathogenic TP53 mutation status.
iPSC-Derived PSEN2 (N141I) Astrocytes and Microglia Exhibit a Primed Inflammatory Phenotype
Authors: Sullivan Michael A. , Lane Samuel D. , Ball Sarah R. , Sunde Margaret , Neely G. Gregory. , Moreno Cesar , Werry Eryn L. , Kassiou Michael
Published: December 2022

Background

Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer’s disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells.

Methods

iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aβ42and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently labelled fibrillar Aβ42.

Results

AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100β and increased secretion and phagocytosis of Aβ42while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells show exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia.

Conclusion

Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a ‘primed’ phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aβ42production and phagocytosis.
Frame Editors for Precise, Template-Free Frameshifting
Authors: Nakade Shota , Nakamae Kazuki , Tang Tzu-Chieh , Yu Dou , Sakuma Tetsushi , Yamamoto Takashi , Lu Timothy K.
Published: December 2022

Efficiency and accuracy are paramount in genome editing. While CRISPR-Cas nucleases are efficient at editing target genes, their accuracy is limited because following DNA cleavage by Cas proteins, error-prone repair mechanisms introduce random mutations. Improving the accuracy of CRISPR-Cas by reducing random repairs using DNA- or RNA-based templates can compromise efficiency. To simultaneously improve both editing efficiency and accuracy, we created a frameshifting genome-editing technology by fusing Cas9 with DNA polymerases. These Frame Editors (FEs) introduce precise and controlled frameshifts into target loci via specific DNA repairs near Cas9-induced cleavage loci. We demonstrate two types of FEs: the insertion-inducing frame editor (iFE) and the deletion-inducing frame editor (dFE). For iFE, DNA polymerase beta (POLB) is fused with Cas9, which increases the frequency of 1-bp insertions. For dFE, T4 DNA polymerase (T4pol) is fused with Cas9, which increases the frequency of 1-bp deletions. Both types of FEs reduce the number of random mutations at target loci compared with Cas9. We show that off-target editing can be reduced by substituting Cas9 with high-fidelity variants, such as HiFi Cas9 or LZ3 Cas9. Thus, FEs can introduce frameshifts into target loci with much improved mutation profiles compared with Cas9 alone and without the requirement for template sequences, offering a new strategy for repairing pathogenic frameshifts.
Generation of adult hippocampal neural stem cells occurs in the early postnatal dentate gyrus and depends on cyclin D2
Authors: Pastor-Alonso Oier , Zahra Anum Syeda , Kaske Bente , García-Moreno Fernando , Tetzlaff Felix , Bockelmann Enno , Grunwald Vanessa , Martin-Suarez Soraya , Riecken Kristoffer , Witte Otto Wilhelm , Encinas Juan Manuel , Urbach Anja
Published: December 2022

In the hippocampus, lifelong neurogenesis is maintained by a pool of multipotent adult neural stem cells (aNSCs) residing in the subgranular zone of the dentate gyrus (DG). Yet, the mechanisms guiding the transition of NSCs from developmental to adult remain unclear. By using nestin-reporter mice deficient for D2, a cyclin expressed mainly postnatally, we show that the aNSC pool is established through D2-dependent proliferation during the first two weeks of life. The absence of D2 allows the normal development of the DG until birth but prevents the postnatal formation of radial glia-like aNSCs. Additionally, retroviral fate mapping demonstrates that aNSCs are born on-site from precursors located in the DG shortly after birth. Altogether, our data suggest that aNSCs are a population distinct from developmental NSCs and thus imply that adult hippocampal neurogenesis is not a mere continuation of development.
Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR
Authors: Obana Nozomu , Takada Hiraku , Crowe-McAuliffe Caillan , Iwamoto Mizuki , Egorov Artyom A. , Wu Kelvin J.Y. , Murina Victoriia , Paternoga Helge , Tresco Ben I.C. , Nomura Nobuhiko , Myers Andrew G. , Atkinson Gemma C. , Wilson Daniel N. , Hauryliuk Vasili
Published: December 2022

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 fromStreptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacteriumNeobacillus vireti) and Clostridia (CplR fromClostridium perfringens,Clostridium sporogenesandClostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate thatC. difficileCplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to theC. difficile630 clinical isolate. Finally, assisted by our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction ofcplRexpression upon an antibiotic challenge.
Isolation and genome characterization of Lloviu virus from Italian Schreibers’ bent-winged bats
Authors: Tóth Gábor E. , Hume Adam J. , Suder Ellen L. , Zeghbib Safia , Ábrahám Ágota , Lanszki Zsófia , Varga Zsaklin , Tauber Zsófia , Földes Fanni , Zana Brigitta , Scaravelli Dino , Scicluna Maria Teresa , Pereswiet-Soltan Andrea , Görföl Tamás , Terregino Calogero , De Benedictis Paola , Garcıa-Dorival Isabel , Alonso Covadonga , Jakab Ferenc , Mühlberger Elke , Leopardi Stefania , Kemenesi Gábor
Published: December 2022

Lloviu cuevavirus (LLOV) was the first identified member ofFiloviridaefamily outside theEbolaandMarburgvirusgenera. A massive die-off of Schreibers’ bent-winged bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its discovery.

Studies with recombinant and wild-type LLOV isolates confirmed the susceptibility of human-derived cell lines and primary human macrophages to LLOV infectionin vitro. Based on these data, LLOV is now considered as a potential zoonotic virus with unknown pathogenicity to humans and bats.

We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding sequence of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences.

The most important achievement of this article is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection. These results further confirms the role of these bats as the host of this virus, possibly throughout their entire geographic range. This is an important result to further understand the role of bats as the natural hosts for zoonotic filoviruses.
Predicting distributed working memory activity in a large-scale mouse brain: the importance of the cell type-specific connectome
Authors: Ding Xingyu , Froudist-Walsh Sean , Jaramillo Jorge , Jiang Junjie , Wang Xiao-Jing
Published: December 2022

Recent advances in connectome and neurophysiology make it possible to probe whole-brain mechanisms of cognition and behavior. We developed a large-scale model of the mouse multiregional brain for a cardinal cognitive function called working memory, the brain’s ability to internally hold and process information without sensory input. The model is built on mesoscopic connectome data for inter-areal cortical connections and endowed with a macroscopic gradient of measured parvalbumin-expressing interneuron density. We found that working memory coding is distributed yet exhibits modularity; the spatial pattern of mnemonic representation is determined by long-range cell type-specific targeting and density of cell classes. Cell type-specific graph measures predict the activity patterns and a core subnetwork for memory maintenance. The model shows numerous self-sustained internal states (each engaging a distinct subset of areas). This work provides a framework to interpret large-scale recordings of brain activity during cognition, while highlighting the need for cell type-specific connectomics.
Multi-omics analysis identifies RFX7 targets involved in tumor suppression and neuronal processes
Authors: Schwab Katjana , Coronel Luis , Riege Konstantin , Sacramento Erika K. , Rahnis Norman , Häckes David , Cirri Emilio , Groth Marco , Hoffmann Steve , Fischer Martin
Published: December 2022

Recurrently mutated in lymphoid neoplasms, the transcription factor RFX7 is emerging as a tumor suppressor. Previous reports suggested that RFX7 may also have a role in neurological and metabolic disorders. We recently reported that RFX7 responds to p53 signaling and cellular stress. Furthermore, we found RFX7 target genes to be dysregulated in numerous cancer types also beyond the hematological system. However, our understanding of RFX7’s target gene network and its role in health and disease remains limited. Here, we generated RFX7 knock-out cells and employed a multi-omics approach integrating transcriptome, cistrome, and proteome data to obtain a more comprehensive picture of RFX7 targets. We identify novel target genes linked to RFX’s tumor suppressor function and underscoring its potential role in neurological disorders. Importantly, our data reveal RFX7 as a mechanistic link that enables the activation of these genes in response to p53 signaling.
Why is the perceptual octave stretched? An account based on mismatched time constants within the auditory brainstem
Authors: de Cheveigné Alain
Published: December 2022

This paper suggests an explanation for listeners’ greater tolerance to positive than negative mistuning of the higher tone within an octave pair. It hypothesizes a neural circuit tuned to cancel the lower tone, that also cancels the higher tone if that tone is in tune. Imperfect cancellation is the cue to mistuning of the octave. The circuit involves two neural pathways, one delayed with respect to the other, that feed a coincidence-sensitive neuron via excitatory and inhibitory synapses. A mismatch between the time constants of these two synapses results in an asymmetry in sensitivity to mismatch. Specifically, if the time constant of thedelayedpathway is greater than that of the direct pathway, there is a greater tolerance topositivemistuning than to negative mistuning. The model is directly applicable to the harmonic octave (concurrent tones), but extending it to the melodic octave (successive tones) requires additional assumptions that are discussed. The paper reviews evidence from auditory psychophysics and physiology in favor – or against – this explanation.
De novo linear phosphorylation site motifs for BCR-ABL kinase revealed by phospho-proteomics in yeast
Authors: Smolnig Martin , Fasching Sandra , Stelzl Ulrich
Published: December 2022

BCR-ABL is the oncogenic fusion product of tyrosine kinase ABL1 and a highly frequent driver of acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML). The kinase activity of BCR-ABL is strongly elevated, however changes of substrate specificity in comparison to wild-type ABL1 kinase are less well characterized. Here, we heterologously expressed full-length BCR-ABL kinases in yeast. We exploited the proteome of living yeast asin vivophospho-tyrosine substrate for assaying human kinases specificity. Phospho-proteomic analysis of ABL1 and BCR-ABL isoforms p190 and p210 yielded a high-confidence dataset of 1127 phospho-tyrosine sites on 821 yeast proteins. We used this data set to generate linear phosphorylation site motifs for ABL1 and the oncogenic ABL1 fusion proteins. The oncogenic kinases yielded a substantially different linear motif when compared to ABL1. Kinase set enrichment analysis with human pY-sites that have high linear motif scores well recalled BCR-ABL driven cancer cell lines from human phospho-proteome data sets.

Highlights

Full-length BCR-ABL kinase expression in yeast

1,127 pY-sites on 821 yeast proteins originating from ABL1 and BCR-ABL kinase activity

Distinct linear kinase motif forABL1 and BCR-ABL p190 and p210 isoforms

Validation of the kinase motifs through KSEA of human cancer cell line phospho-proteome data

Significance

The fusion protein BCR-ABL is a predominant oncogene in leukemia observed at very high frequency in chronic myeloid leukemia (BCR-ABL p210) and B-cell acute lymphoid (BCR-ABL p190) leukemia. In both cases the malignant transformation is reliant on elevated tyrosine kinase activity, however ABL1 kinase activation as such is not sufficient. At least in part, leukemias may therefore be driven by a specificity switch of the fusion kinase in comparison to the wild-type ABL1.

We investigated BCR-ABL kinase substrates in yeast through phospho-proteomics and established linear kinase phosphorylation-motifs for the full length BCR-ABL isoforms p210 and p190. These novel motifs differ substantially from the corresponding specificity determinants of wild-type ABL1 kinase, providing direct experimental evidence for altered phosphorylation-specificity of the oncogenic kinase fusion proteins.
Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy
Authors: Last Mart G. F. , Tuijtel Maarten W. , Voortman Lenard M. , Sharp Thomas H.
Published: December 2022

Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy (FM) are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude, and demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated microscopy.
Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging
Authors: Vandiver Amy R. , Hoang Austin N. , Herbst Allen , Lee Cathy C. , Aiken Judd M. , McKenzie Debbie , Timp Winston , Wanagat Jonathan
Published: December 2022

Background

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency.

Results

We employed nanopore Cas9-targed sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints.

Conclusions

NCATS-mtDNA sequencing allows identification of mtDNA deletions on a single molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.
Transcriptional activity mediated by β-CATENIN and TCF/LEF family members is completely dispensable for survival of multiple human colorectal cancer cell lines
Authors: Fröhlich Janna , Rose Katja , Hecht Andreas
Published: December 2022

Unrestrained transcriptional activity of β-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, theTCF7L2gene carries inactivating mutations in about 10 % of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family members, or rather lose addiction to β-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or β-CATENIN expression. Viability of these cells demonstrates complete β-CATENIN- and TCF/LEF-independence, albeit one β-CATENIN-deficient cell line eventually became senescent. Absence of TCF/LEF proteins and β-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/β-CATENIN signaling in the healthy intestine. Despite this common phenotype, β-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between β-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, β-CATENIN and TCF7L2 control sizeable fractions of their target genes independently from each other. The observed divergence of β-CATENIN and TCF7L2 transcriptional programs, and the finding that neither β-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets.
Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane
Authors: Shi Wei , Cai Yongfei , Zhu Haisun , Peng Hanqin , Voyer Jewel , Rits-Volloch Sophia , Cao Hong , Mayer Megan L. , Song Kangkang , Xu Chen , Lu Jianming , Zhang Jun , Chen Bing
Published: December 2022

Entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells depends on refolding of the virus-encoded spike protein from a prefusion conformation, metastable after cleavage, to a lower energy, stable postfusion conformation. This transition overcomes kinetic barriers for fusion of viral and target cell membranes. We report here a cryo-EM structure of the intact postfusion spike in a lipid bilayer that represents single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membraneinteracting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.
Neuro-immune modulation of cholinergic signaling in an addiction vulnerability trait
Authors: Carmon Hanna , Haley Evan C. , Parikh Vinay , Tronson Natalie C. , Sarter Martin
Published: December 2022

Sign-tracking (ST) describes the propensity to approach and contact a Pavlovian reward cue. By contrast, goal-trackers (GTs) respond to such a cue by retrieving the reward. These behaviors index the presence of opponent cognitive-motivational traits, with STs exhibiting attentional control deficits, behavior dominated by incentive motivational processes, and vulnerability for addictive drug taking. Attentional control deficits in STs were previously attributed to attenuated cholinergic signaling, resulting from deficient translocation of intracellular choline transporters (CHTs) into synaptosomal plasma membrane. Here we investigated a post-translational modification of CHTs – poly-ubiquitination - and tested the hypothesis that elevated cytokine signaling in STs contributes to CHT modification. We demonstrated that intracellular CHTs, but not plasma membrane CHTs, are highly ubiquitinated in male and female sign-tracking rats when compared with GTs. Moreover, levels of cytokines measured in cortex and striatum, but not spleen, were higher in STs than in GTs. Activation of the innate immune system by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) elevated ubiquitinated CHT levels in cortex and striatum of GTs only, suggesting ceiling effects in STs. In spleen, LPS increased levels of most cytokines in both phenotypes. In cortex, LPS particularly robustly increased levels of the chemokines CCL2 and CXCL10. Phenotype-specific increases were restricted to GTs, again suggesting ceiling effects in STs. These results indicate that interactions between elevated brain immune modulator signaling and CHT regulation are essential components of the neuronal underpinnings of the addiction vulnerability trait indexed by sign-tracking.

Significance Statement

Sign-tracking rats (STs) have emerged as a fruitful animal model for determining the neuro-behavioral foundations, including the cholinergic-attentional control deficits, which heighten the risk for the manifestation of addictive behaviors. The results from the present experiments suggest that, in STs, elevated levels of neuro-immune signaling interact with a post-translational modification of choline transporters that yields a relatively low transporter capacity. Furthermore, the effects of activation of the innate immune system, by administrating the endotoxin lipopolysaccharide, mechanistically supported such an interaction. The results extend research on the neuronal vulnerabilities for addictive behaviors to the role of neuro-immune signaling and suggests a new role of neuro-immune modulators in influencing complex cognitive-motivational traits.
The 3D chromatin landscape of rhabdomyosarcoma
Authors: Wang Meng , Sreenivas Prethish , Sunkel Benjamin D. , Wang Long , Ignatius Myron , Stanton Benjamin Z.
Published: December 2022

Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with a lack of precision therapy option for patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depthin situHi-C in representative cell lines and patient-derived xenografts to understand chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative rhabdomyosarcoma (FN-RMS). We have generated spike-inin situHi-C chromatin interaction maps for the most common FP-RMS and FN-RMS cell lines, and compared our data with patient derived xenograft (PDX) models. In our studies we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains, and unique patterns of structural variation. Our comprehensive analysis provides high-depth chromatin interactivity maps for contextualizing gene regulation events identification of functionally critical chromatin domains in RMS.

HIGHLIGHTS

PAX3-FOXO1 and MYOD localize in both A- and B-compartments

Conserved mechanisms dictate CTCF orientation at TAD boundaries in RMS

Differential TADs in each RMS subtype encompass tumor-specific genes

Neo-TADs are formed from SV events in each subtype of RMS

Both major RMS subtypes have structural variation that is identifiable from Hi-C

Distinct mechanisms can produce the major fusion alleles in rhabdomyosarcoma

PAX3-FOXO1 and MYOD genomic binding is more enriched at regions with CNV
Characterization of a strain-specific CD-1 reference genome reveals potential inter- and intra-strain functional variability
Authors: Jung Yoon-Hee , Wang Hsiao-Lin , Ali Samir , Corces Victor G. , Kremsky Isaac
Published: December 2022

Background

CD-1 is an outbred mouse stock that is frequently used in toxicology, pharmacology, and fundamental biomedical research. Although inbred strains are typically better suited for such studies due to minimal genetic variability, outbred stocks confer practical advantages over inbred strains, such as improved breeding performance and low cost. Knowledge of the full genetic variability of CD-1 would make it more useful in toxicology, pharmacology, and fundamental biomedical research.

Results

We performed deep genomic DNA sequencing of CD-1 mice and used the data to identify genome-wide SNPs, indels, and germline transposable elements relative to the mm10 reference genome. We used multiple genome-wide sequencing data types and previously published CD-1 SNPs to validate our called variants. We used the called variants to construct a strain-specific CD-1 reference genome, which we show can improve mappability and reduce experimental biases from genome-wide sequencing data derived from CD-1 mice. Based on previously published ChIP-seq and ATAC-seq data, we find evidence that genetic variation between CD-1 individuals can lead to alterations in transcription factor binding. We also identified a number of variants in the coding region of genes which could have effects on splicing and translation of genes.

Conclusions

We have identified millions of previously unidentified CD-1 variants with the potential to confound studies involving CD-1. We used the identified variants to construct a CD-1-specific reference genome, which can improve accuracy and reduce bias when aligning genomics data derived from CD-1 individuals.
Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis
Authors: VanArsdale Eric , Navid Ali , Chu Monica J. , Halvorsen Tiffany M. , Payne Gregory F. , Jiao Yongqin , Bentley William E. , Yung Mimi C.
Published: December 2022

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by “programming” cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon ofEscherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including “monoculture” and “transmitter-receiver” models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the “collective” attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.
Modified bronchoscopy masks mitigate aerosols during gastroscopies
Authors: Phillips Frank , Crowley Jane , Warburton Samantha , Gordon George S.D. , Parra-Blanco Adolfo
Published: December 2022

Digestive endoscopy has been proven to produce aerosols (1–3). This represents a risk of infection by COVID-19 and other airborne viruses. A number of protective barriers have been proposed to minimise that risk. Continuous suction of the oral cavity (1), shielding barriers (4,5), masks (6,7), and increasing the distance between patient and endoscopist (8) have been proposed as methods to reduce the exposure of endoscopists and endoscopy staff to aerosols. Here, we present a study that uses modified bronchoscopy masks (Explorer endoscopy facemask, Intersurgical Ltd., United Kingdom) to attenuate aerosol production at the patient’s mouth (bare mask shown in Fig. 1a and in use during an upper GI endoscopy in Fig. 1b). We find that this approach offers 47% (p=0.01) reduction in particle count for particles <5μm in diameter (i.e. aerosols), which are known to spread SARS-CoV-2.
Quality and safety for the public through domiciliary nursing in Italy: a multicentre cross-sectional descriptive observational study (The AIDOMUS-IT Protocol)
Authors: Bagnasco Annamaria , Alvaro Rosaria , Lancia Loreto , Manara Duilio Fiorenzo , Rasero Laura , Rocco Gennaro , Maurizio Zega , Mazzoleni Beatrice , Sasso Loredana
Published: December 2022

Introduction

The development of this study protocol occurred in conjunction with the new Regulation defining models and standards for the development of community care in the National Health Service (Ministerial Decree n. 77-2022) with the redefinition of care in the community. Considered the increase in the elderly population, in the complexity of care and the workload for home care, it is important and urgent to describe the work environment and the Italian community care reality. The main objective of this study is to evaluate the characteristics of nursing care and the quality of home care in the community in Italy.

Methods

This is a cross-sectional descriptive observational study using the survey method. The study protocol uses quantitative data from three sources: 1) primary data on organizational characteristics, professional satisfaction, intention to leave home care services, and burnout; 2) secondary data on the experience of patients and their informal carers; 3) data on improper access to the emergency department, readmission to hospital, comorbidities, services offered, and user level of autonomy, and main and secondary diagnoses. Data will be analyzed using descriptive and inferential statistics.

Conclusions

The systematic analysis of the different national community care contexts will enable to increase the knowledge and awareness of the need for community health care. The availability of specific data will promote and support the role of the family and community nurse in district health care contexts, to increase the quality of care perceived by patients and their families.