bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hastened fusion-dependent endosomal escape improves activity of delivered enzyme cargo
Authors: Vázquez Maldonado Angel Luis , Chen Teresia , Rodriguez Diego , Zoltek Madeline , Schepartz Alanna 
Published: September 2024

AbstractThere is enormous interest in strategies to efficiently traffic biologics–proteins, nucleic acids, and complexes thereof–into the mammalian cell cytosol and internal organelles. Not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment can denature and/or degrade internalized material. ZF5.3 is a compact, stable, rationally designed mini-protein that efficiently escapes intact from late endocytic vesicles, with or without covalently linked protein cargo. Here, using insights from mechanistic studies on the pathway of endosomal escape and classic principles of zinc(II) bioinorganic chemistry, we re-designed the sequence of ZF5.3 to successfully alter the timing (but not the efficiency) of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity of a pH-sensitive enzyme upon arrival in the cytosol. Delivery of the pH-sensitive enzyme dihydrofolate reductase (DHFR) with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of AV5.3-DHFR upon delivery is sufficient to rescue a genetic DHFR deletion in CHO cells. This work provides evidence that programmed trafficking through the endosomal pathway is a viable strategy for the efficient cytosolic delivery of therapeutic proteins.Abstract Figure
Lysophospholipid headgroup size, and acyl chain length and saturation differentially affect vacuole acidification, Ca2+transport, and fusion
Authors: Zhang Chi , Feng Yilin , Calderin Jorge D. , Balutowski Adam , Ahmed Razeen , Knapp Charlie , Fratti Rutilio A. 
Published: September 2024

ABSTRACTSNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipid composition impacts fusion through direct protein lipid interactions or through modulating the physical properties of membranes at the site of contact, including the induction of positive curvature by lysophospholipids (LPLs). The degree of positive curvature induced is due to the length and saturation of the single acyl chain in addition to the size of the head group. Here we examined how yeast vacuole fusion and ion transport were differentially affected by changes in lysolipid properties. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons all inhibited fusion with IC50values ranging from ∼40-120 µM. The monounsaturation of LPC-18:1 had no effect when compared to its saturated counterpart LPC-18:0. On the other hand, head group size played a more significant role in blocking fusion as lysophosphatidic acid (LPA)-18:1 failed to fully inhibit fusion. We also show that both Ca2+uptake and SNARE-dependent Ca2+efflux was sensitive to changes in the acyl chain length and saturation of LPCs, while LPA only affected Ca2+efflux. Finally, we tested these LPLs on vacuole acidification by the V-ATPase. This showed that LPC-18:0 could fully inhibit acidification whereas other LPCs had moderate effects. Again, LPA had no effect. Together these data suggest that the effects of LPLs were due to a combination of head group size and acyl chain length leading to a range in degree of positive curvature.
irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA
Authors: Ducoli Luca , Zarnegar Brian J. , Porter Douglas F. , Meyers Robin M. , Miao Weili , Riley Nicholas M. , Srinivasan Suhas , Jackrazi Leandra V. , Yang Yen-Yu , Li Zhouxian , Wang Yinsheng , Bertozzi Carolyn R. , Flynn Ryan A. , Khavari Paul A. 
Published: September 2024

AbstractRNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation1-3. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease1,4,5, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation6was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
Loss of Ing4 enhances hematopoietic regeneration in multipotent progenitor cells
Authors: Anderson Georgina A. , Hernandez Marco , Quinde Carlos Alfaro , Thompson Zanshé , Rodriguez Melanie , Binder-Blaser Vera , Taylor Alison M. , Kathrein Katie L. 
Published: September 2024

SummaryDespite its critical role in survival, many aspects of hematopoiesis remain unresolved. In the classical model of the hematopoietic program, quiescent hematopoietic stem cells (HSCs) sit at the top of the hematopoietic hierarchy, with the ability to self-renew and differentiate as needed. HSCs give rise to more proliferative progenitor cells, which possess multipotent potential, but have largely or completely lost self-renewal capabilities. Here, we have identified the tumor suppressor, Inhibitor of Growth 4 (Ing4), as a critical regulator of multipotent progenitor (MPP) homeostasis. In the absence of Ing4, we show that MPPs express a transcriptional program of hematopoietic activation, yet they remain quiescent with low levels of reactive oxygen species. Functionally, Ing4-deficient MPPs are capable of robust regeneration following competitive bone marrow transplantation, resulting in substantially higher blood chimerism compared to wild-type MPPs. These data suggest Ing4 deficiency promotes a poised state in MPPs, quiescent, but transcriptionally primed for activation, and capable of converting the poised state into robust repopulation upon stress. Our model provides key tools for further identification and characterization of pathways that control quiescence and regeneration in MPPs.
Photosymbiosis Shaped Animal Genome Architecture and Gene Evolution as Revealed in Giant Clams
Authors: Li Ruiqi , Leiva Carlos , Lemer Sarah , Kirkendale Lisa , Li Jingchun 
Published: October 2024

AbstractSymbioses are major drivers of organismal diversification and phenotypic innovation. However, how long-term symbioses shape whole genome evolution in metazoans is still underexplored. Here, we used a giant clam (Tridacna maxima) genome to demonstrate how symbiosis has left complex signatures in an animal’s genome. Giant clams thrive in oligotrophic waters by forming a remarkable association with photosymbiotic dinoflagellate algae. Genome-based demographic inferences uncovered a tight correlation betweenT. maximaglobal population change and major paleoclimate and habitat shifts, highlighting how abiotic and biotic factors dictateT. maximamicroevolution. Comparative analyses revealed unique symbiosis-driven genomic features, including expansion and contraction of immunity-related gene families and a large proportion of lineage-specific genes. Strikingly, about 70% of the genome is composed of repetitive elements, especially transposable elements, most likely resulting from a symbiosis-adapted immune system. This work greatly enhances our understanding of genomic drivers of symbiosis that underlie metazoan evolution and diversification.
Investigating the integration of sustainable food initiatives in healthcare institutions in Ontario, Canada: A grey literature scoping review protocol
Authors: Blank Lisa L. , Milano Alyssa D. , Andrade Lesley , Kirkpatrick Sharon I. 
Published: September 2024

AbstractIntroductionThe climate emergency and other sustainability challenges interact to threaten human and planetary health. Efforts to improve the sustainability of food initiatives within healthcare institutions could mitigate these threats by addressing the four pillars of sustainability: health, social, economic, and environmental. Understanding current initiatives to incorporate sustainability into food programs and the sustainability pillars that guide those initiatives is important to inform priorities for action.This scoping review was undertaken to investigate the extent to which major healthcare institutions in Ontario, Canada have publicly committed to, discussed, planned, and/or implemented sustainable food initiatives.Methods and AnalysisSteps are based on guidance from the Joanna Briggs Institute and Arksey & O’Malley. First, the current strategic plans of 57 healthcare institutions in Ontario, Canada, will be retrieved from their websites and used to examine whether they include any commitments to or discussion, planning, and/or implementation of relevant initiatives. The healthcare institution websites, along with those of selected sustainability organizations, will be searched for grey literature from 2015 to 2024 describing sustainable food initiatives within these institutions. Documents will be screened for eligibility by two researchers. Data related to the incorporation of sustainable food into institutional food programs, and the sustainability pillars addressed, will be extracted by one researcher, with 10% of entries verified by a second researcher. The data will be synthesized to summarize publicly reported progress toward integrating sustainable food into healthcare institutions.Ethics and DisseminationThis review will use publicly available grey literature with no expectation of privacy and no research participants; therefore, no ethics clearance is required.Results will be shared with stakeholders in sustainability organizations, as well as at relevant conferences and in peer-reviewed journals, such as the Healthy Cities Conference and the Journal of Canadian Food Studies.This protocol is registered on the Open Science Framework and can be accessed at the following URL:https://doi.org/10.17605/OSF.IO/CU9P6
Mapping the Mind in the Virtual Metaverse: An Initial In-Depth Thematic Exploration of Youth Mental Health within VRChat
Authors: Um Tevin S , Ownby Raymond L , Chou Shinnyi 
Published: October 2024

AbstractBackgroundVRChat is a virtual reality (VR) social video game where users play a personalized avatar and socialize with others in community-built worlds. Its immersive nature and diverse community may impact youth users’ mental health, yet no research has explored this. This study is a thematic exploration of VRChat’s effect on youth mental health.MethodsSemi-structured interviews were conducted with 20 active VRChat users aged 18-24 years. Interviews were transcribed and analyzed using grounded theory principles to identify themes related to VRChat use and mental health.ResultsNine major themes emerged: Frequency of use; purpose of use; positive effects on mental health; negative effects on mental health; social connection and COVID-19; anonymity; cyberbullying and toxic behaviors; addiction; and recommendations for a safer environment. Positive effects identified included enhanced social connection, creative expression, and self-confidence. Negative effects included addiction, cyberbullying, predatory behaviors, and worsening of existing mental illnesses. The anonymity of VRChat created a paradoxical environment that can foster both genuine self-expression and toxic behaviors. During the COVID-19 pandemic, VRChat served as an important substitute for real-life social interactions. Participants also provided recommendations for developers, parents, and clinicians to promote a safer VRChat experience.ConclusionThe immersive and interactive social experience offered by VRChat is conducive for positive effects on youth users’ mental health, such as social connectivity and self-expression. However, it also presents risks such as addiction and toxic behaviors. Given the interactive nature of VRChat, further research may elucidate its potential as a psychotherapeutic intervention for youth mental health.
A repository of Ogden syndrome patient derived iPSC lines and isogenic pairs by X-chromosome screening and genome-editing
Authors: Wesely Josephine , Rusielewicz Tom , Chen Yu-Ren , Hartley Brigham , McKenzie Dayna , Yim Matthew K. , Maguire Colin , Bia Ryan , Franklin Sarah , Makwana Rikhil , Marchi Elaine , Nikte Manali , Patil Soha , Sapar Maria , Moroziewicz Dorota ,   NYSCF Global Stem Cell Array® Team, Bauer Lauren , Lee Jeannie T. , Monsma Frederick J. , Paull Daniel , Lyon Gholson J. 
Published: September 2024

AbstractAmino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting 80% of cytosolic proteins in humans. The human essential gene,NAA10,encodes the enzyme NAA10, as the catalytic subunit for the N-terminal acetyltransferase A (NatA) complex, including the accessory protein, NAA15. The first human disease directly involvingNAA10was discovered in 2011, and it was named Ogden syndrome (OS), after the location of the first affected family residing in Ogden, Utah, USA. Since that time, other variants have been found inNAA10andNAA15.Here we describe the generation of 31 iPSC lines, with 16 from females and 15 from males. This cohort includes CRISPR-mediated correction to the wild-type genotype in 4 male lines, along with editing one female line to generate homozygous wild-type or mutant clones. Following the monoclonalizaiton and screening for X-chromosome activation status in female lines, 3 additional pairs of female lines, in which either the wild type allele is on the active X chromosome (Xa) or the pathogenic variant allele is on Xa, have been generated. Subsets of this cohort have been successfully used to make cardiomyocytes and neural progenitor cells (NPCs). These cell lines are made available to the community via the NYSCF Repository.
Optimal Frequency for Seizure Induction with Electroconvulsive Therapy and Magnetic Seizure Therapy
Authors: Peterchev Angel V. , Deng Zhi-De , Sikes-Keilp Christopher , Feuer Elyssa C. , Rosa Moacyr A. , Lisanby Sarah H. 
Published: October 2024

AbstractElectroconvulsive therapy (ECT) and magnetic seizure therapy (MST) are effective in the treatment of medication-resistant depression. Determining the stimulus frequency resulting in the lowest seizure threshold could produce fewer adverse effects by reducing the overall stimulus intensity. To determine the optimal frequency for seizure induction, four male rhesus macaques were titrated with an increasing number of pulses at fixed frequencies ranging from 5 to 240 pulses per second (pps) using ultrabrief-pulse right-unilateral ECT and circular-coil-on-vertex MST. The seizure threshold dependence on stimulus frequency was similar for ECT and MST. While higher frequencies required progressively shorter trains to induce a seizure, the middle frequency range was associated with the fewest pulses (and hence the least charge and energy), with a minimum at 16 pps and similarly low thresholds for 10 and 25 pps. The number of pulses at seizure threshold increased markedly at lower and higher frequencies. The lowest stimulus frequencies, 5 and 10 pps, were associated with the greatest ictal power measured by electroencephalography. While this study did not assess efficacy or side effects, the results highlight the significance of stimulus frequency for seizure induction, suggest efficient titration schedules that minimize exposure to the electrical stimulus, and can inform studies to assess the impact on clinical outcomes.
Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting
Authors: Alsayyah Cynthia , Rodrigues Emmanuel , Hach Julia , Renne Mike F. , Ernst Robert 
Published: October 2024

AbstractLarge unilamellar vesicles (LUVs) are popular membrane models for studying the impact of lipids and bilayer properties on the structure and function of individual membrane proteins. The functional reconstitution of transmembrane in liposomes can be challenging, especially, if the hydrophobic thickness of the protein does not match the thickness of the surrounding lipid bilayer. During the reconstitution procedure Such hydrophobic mismatch causes low yields and protein aggregation, which are exacerbated in sterol-rich membranes featuring low membrane compressibility. Here, we explore new approaches to reversibly tune membrane sterol contents proteoliposomes after their formation. Both cholesterol delivery and extraction are mediated by methyl-β-cyclodextrin in a dialysis setting, which maintains (proteo)liposomes in a confined compartment. This makes it possible to reversibly tune the cholesterol level without losing membrane material simply by placing the dialysis cassette in a new bath containing either empty or cholesterol-loaded methyl-β-cyclodextrin. Cholesterol delivery and removal is monitored with the solvatochromic probe C-Laurdan, which reports on lipid packing. Using Förster-resonance energy transfer, we show that cholesterol delivery to proteoliposomes induces the oligomerization of a membrane property sensor, while the subsequent removal of cholesterol demonstrates the full reversibility. We propose that tuning membrane compressibility by methyl-β-cyclodextrin-meditated cholesterol delivery and removal in a dialysis setup provides a new handle to study its impact on membrane protein structure, function, and dynamics.Statement of significanceGenerating complex, sterol-rich, biomimetic membranes for studying the structure and function of reconstituted membrane proteins is challenging. As an important step towards asymmetric, sterol-rich, complex model membrane systems, we have established a procedure to control the membrane sterol level of liposomes and proteoliposomes using methyl-β-cyclodextrin in a dialysis setup. We demonstrate the feasibility of this approach by C-Laurdan and dehydroergosterol fluorescence spectroscopy and gain control over the membrane sterol content. We explore several parameters that affect the rate of cholesterol delivery and show that the oligomerization of a membrane property sensor, which is on the unfolded protein response sensor protein Ire1, is controlled by the sterol content of the surrounding lipid bilayer.
Real-time affinity measurements of proteins synthesized in cell-free lysate using fluorescence correlation spectroscopy
Authors: Liu Chao , Hoang-Phou Steven A. , Ye Congwang , Laurence Emma J. , Laurence Matthew J. , Fong Erika J. , Hammond Nikki M. , Dillon Vannest B. , Watkins Nicholas N. , Laurence Ted A. , Coleman Matthew A. 
Published: October 2024

ABSTRACTRapid, high throughput measurements of biomolecular interactions are essential across medicine and bioscience. Traditional methods for affinity-screening proteins require a long and costly process involving cell-based expression, purification, and titration of multiple concentrations to arrive at a binding curve. In contrast, we have developed a fast and simple approach that yields a wealth of information about the expression of the protein and its binding characteristics, all in a “one-pot reaction” and done in under several hours without the need for protein purification. The method uses cell-free protein synthesis to produce the protein of interest in the presence of its binding partner, while simultaneously using fluorescence correlation spectroscopy (FCS) to measure the increasing concentration of the protein and its binding to the binding partner. We characterize the sensitivity limits of this method by measuring the binding between the green fluorescent protein (GFP) and a low picomolar-affinity anti-GFP antibody and found that we can quantifyKDs down to the high picomolar to low-nanomolar range. We further demonstrate the method in a potentially ultrahigh-throughput sample format, in which FCS measurements are collected inside microcapsules. This work lays the foundation for a platform aimed at production and in situ affinity screening of thousands of different proteins.
Transcriptomic signatures of host immune responses in aphthous ulcers, the earliest lesions of Crohn’s disease, suggest that a bacterial invasive challenge, rather than global dysbiosis, is the initiating factor
Authors: Whiley Phillip J , Dixit Ojas V A , Gupta Mukta Das , Patel Hardip , Zhao Guoyan , Connor Susan J , Summers Kim M , Hume David A , Pavli Paul , O’Brien Claire L 
Published: September 2024

AbstractCrohn’s disease is a chronic, transmural inflammatory disease of the human gut. Changes in the faecal microbial composition and a reduction in species diversity (dysbiosis) are consistent features in studies of Crohn’s disease patients, but whether dysbiosis is a cause or consequence of inflammation remains unresolved. Genetic susceptibility also plays a role in the development of Crohn’s disease and has been linked to genes involved in recognition of intestinal bacteria by cells of the mononuclear phagocyte system. The earliest visible lesions in Crohn’s disease are aphthous ulcers, overlying Peyer’s patches and lymphoid follicles. To identify mechanisms underlying the earliest stages of disease initiation we compared gene expression in aphthous ulcers, Peyer’s patches, inflamed and endoscopically normal mucosa from patients and controls using total RNA-seq.The resulting data was subjected to network analysis to identify co-regulated gene expression signatures of cell types and processes. These results were compared to recent single cell RNA-seq analysis of intestinal macrophages in normal and diseased mucosa. The analysis of aphthous ulcers revealed signatures of epithelial stress and antimicrobial defence, plasma cell activation and immunoglobulin production, monocyte recruitment, inflammatory gene expression and induction of interferon-γ and downstream target genes. These signatures were not present in the normal appearing mucosa adjacent to aphthous ulcers which were similar to healthy control mucosa.We conclude that the initial lesion in Crohn’s disease arises from an invasive bacterial challenge leading to intense activation of multiple host defence pathways rather than the breakdown of epithelial barrier integrity and widespread bacterial translocation.