bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Protein Interaction Networks of Catalytically-Active and Catalytically-Inactive PqsE in Pseudomonas aeruginosa
Authors: Taylor Isabelle R. , Murray-Nerger Laura A. , Cristea Ileana M. , Bassler Bonnie L. 
Published: June 2022

ABSTRACTPseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor, RhlR, an interaction that enhances affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, PqsE virulence function can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing eGFP-PqsE fusions to define the protein interactomes of wildtype PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wildtype PqsE, while not forming associations with PqsE(D73A). In the ΔrhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase to RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing.IMPORTANCEPseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including in immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins: PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates that will be investigated for potential roles in pathogenesis.
Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1
Authors: Nicoli Alessandro , Haag Franziska , Marcinek Patrick , He Ruiming , Kreißl Johanna , Stein Jörg , Marchetto Alessandro , Dunkel Andreas , Hofmann Thomas , Krautwurst Dietmar , Di Pizio Antonella 
Published: June 2022

ABSTRACTWith approximately 400 encoding genes in humans, odorant receptors (ORs) are the largest subfamily of class A G protein-coupled receptors (GPCRs). Despite its high relevance and representation, the odorant-GPCRome is structurally poorly characterized: no experimental structures are available, and the low sequence identity of ORs to experimentally solved GPCRs is a significant challenge for their modeling. Moreover, the receptive range of most ORs is unknown. The odorant receptor OR5K1 was recently and comprehensively characterized in terms of cognate agonists. Here we report two additional agonists and functional data of the most potent compound on two mutants, L1043.32and L2556.51. Experimental data was used to guide the investigation of the binding modes of OR5K1 ligands into the orthosteric binding site using structural information from AI-driven modeling, as recently released in the AlphaFold Protein Structure Database, and from homology modeling. Induced-fit docking simulations were used to sample the binding site conformational space for ensemble docking. Mutagenesis data guided side chain residue sampling and model selection. We obtained models that could better rationalize the different activity of active (agonist) versus inactive molecules with respect to starting models, and also capture differences in activity related to minor structural differences. Therefore, we provide a model refinement protocol that can be applied to model the orthosteric binding site of ORs as well as that of GPCRs with low sequence identity to available templates.
Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors
Authors: Prasad Kirti , Devaraju Nivedhitha , George Anila , Ravi Nithin Sam , Mahalingam Gokulnath , Rajendiran Vignesh , Panigrahi Lokesh , Venkatesan Vigneshwaran , Lakhotiya Kartik , Moorthy Yogapriya , Pai Aswin Anand , Nakamura Yukio , Kurita Ryo , Balasubramanian Poonkuzhali , Thangavel Saravanabhavan , Velayudhan Shaji R , Marepally Srujan , Srivastava Alok , Mohankumar Kumarasamypet M 
Published: June 2022

Abstractβ-thalassemia and HbE result from mutations in the β-globin locus that impedes the production of functional β-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of β-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.
Rapid Activation of Hematopoietic Stem Cells
Authors: Thapa Roshina , Elfassy Erez , Olender Leonid , Sharabi Omri , Gazit Roi 
Published: June 2022

SummaryAdult Hematopoietic Stem Cells (HSCs) in the bone marrow (BM) are quiescent. Following perturbations, such as blood loss or infection, HSCs may undergo activation to accelerate the production of needed effector blood and immune cells. Surprisingly, little is known about the earliest stages of HSC activation. We utilize surface markers of HSC activation, CD69, and CD317, revealing response as early as 2 h after stimulation. The dynamic expression of HSC activation markers varies between viral-like (poly-Inosinic-poly-Cytidylic) or bacterial-like (Lipopolysaccharide) immune stimuli. We further quantify the dose response, demonstrating a low-threshold and similar sensitivity of HSCs and progenitors in the BM. Finally, we find a positive correlation between the expression of surface activation markers and early exit from quiescence into proliferation. Our data show that the premier response of adult stem cells to immune stimulation is rapid, sensitive, and directly leads to proliferation.
Investigation of nano- and micro-domains formed by ceramide-1-phosphate in freestanding lipid bilayers
Authors: Drabik Dominik , Czogalla Aleksander 
Published: June 2022

ABSTRACTBiological membranes are known for their complex nature and formation of domains is crucial for proper execution of multiple cellular processes. Such domains, mostly due to their nanoscale character, are rarely studied since implementation of techniques for their quantitative investigation is difficult. In this article we introduce spot-variation z-scan FCS implemented for the first time for artificial lipid vesicles of defined lipid composition. We used this approach to investigate behaviour of different species of ceramide-1-phosphate within membranes. We were able to provide quantitative description of lipid domains generated by ceramide-1-phosphate in nano and microscale. Aligning these results with in silico studies helped us to validate the approach and draw conclusions on complex behaviour of signalling lipids within biological membranes.
Plant Intron Splicing Efficiency Database (PISE): exploring splicing of ∼1,650,000 introns in Arabidopsis, maize, rice and soybean from ∼57,000 public RNA-seq libraries
Authors: Zhang Hong , Jia Jinbu , Zhai Jixian 
Published: June 2022

ABSTRACTIntron retention is the most common form of alternative splicing events in plants and plays a crucial role in plants responding to environmental signals. Despite a large number of RNA-seq libraries from different treatments and genetic mutants stored at public domains, a resource for querying the intron splicing ratio of individual introns is still missing. Here, we established the first-ever large-scale splicing efficiency database to date in any organism. Our database includes more than 57,000 plant public RNA-seq libraries, including 25,283 from Arabidopsis, 17,789 from maize, 10,710 from rice, and 3,974 from soybean, and in total covers more than 1.6 million introns in these four species. In addition, we have manually curated and annotated all the mutant-and treatment-related libraries as well as their matched controls included in our library collection, and added graphics to display intron splicing efficiency across different tissues, developmental stages, and stress-related conditions. The result is a large collection of 3,313 treatment conditions and 3,594 genetic mutants for discovering the differentially regulated splicing efficiency. Our online database can be accessed at https://plantintron.com/.
A Parabrachial to Hypothalamic Pathway Mediates Defensive Behavior
Authors: Wang Fan , Chen Yuge , Lin Yuxin , Wang Xuze , Li Kaiyuan , Han Yong , Wu Jintao , Shi Xingyi , Zhu Zhenggang , Long Chaoying , Hu Xiaojun , Duan Shumin , Gao Zhihua 
Published: June 2022

AbstractDefensive behaviors are critical for animal’s survival. Both the paraventricular nucleus of the hypothalamus (PVN) and the parabrachial nucleus (PBN) have been shown to be involved in defensive behaviors. However, whether there are direct connections between them to mediate defensive behaviors remains unclear. Here, by retrograde and anterograde tracing, we uncover that cholecystokinin (CCK)-expressing neurons in the lateral PBN (LPBCCK) directly project to the PVN. Byin vivofiber photometry recording, we found that LPBCCKneurons actively respond to various threat stimuli. Selective photoactivation of LPBCCKneurons promotes aversion and defensive behaviors. Conversely, photoinhibition of LPBCCKneurons attenuates rat or looming stimuli-induced flight responses. Optogenetic activation of LPBCCKaxon terminals within the PVN or PVN glutamatergic neurons promote defensive behaviors. Whereas chemogenetic and pharmacological inhibition of local PVN neurons prevent LPBCCK-PVN pathway activation-driven flight responses. These data suggest that LPBCCKneurons recruit downstream PVN neurons to actively engage in flight responses. Our study identifies a previously unrecognized role for the LPBCCK-PVN pathway in controlling defensive behaviors.
Brain Atlas for Glycoprotein Hormone Receptors at Single-Transcript Level
Authors: Ryu Vitaly , Gumerova Anisa , Korkmaz Funda , Kang Seong Su , Katsel Pavel , Miyashita Sari , Kannangara Hasni , Cullen Liam , Chan Pokman , Kuo Tanchun , Padilla Ashley , Zaidi Samir , Kim Se-Min , New Maria I. , Rosen Clifford J. , Goosens Ki A. , Frolinger Tal , Haroutunian Vahram , Ye Keqiang , Lizneva Daria , Davies Terry F. , Yuen Tony , Zaidi Mone 
Published: June 2022

ABSTRACTThere is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHRs, LHCGRs and FSHRs. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and sub–nuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and sub–nuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHRs in human brain samples, where transcripts were co–localized in MALAT1–positive neurons. In addition, we show high expression for all three receptors in the ventricular region—with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. TSHRs were expressed specifically in tanycytes. In contrast, Fshrs were localized to NeuN–positive neurons in the granular layer of the dentate gyrus in murine and human brain—both are Alzheimer’s disease vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.
Acute expression of human APOBEC3B in mice causes lethality associated with RNA editing
Authors: Alonso de la Vega Alicia , Temiz Nuri Alpay , Tasakis Rafail , Somogyi Kalman , Reuveni Eli , Ben-David Uri , Stenzinger Albrecht , Poth Tanja , Papavasiliou Nina , Harris Reuben S. , Sotillo Rocio 
Published: June 2022

AbstractRNA editing has been described to promote heterogeneity leading to the development of multiple disorders including cancer. The cytosine deaminase APOBEC3B is known to fuel tumor evolution through DNA mutagenesis, but whether it may also function as an RNA editing enzyme has not been studied. Here, we engineered a novel doxycycline-inducible mouse model of human APOBEC3B-overexpression to understand the impact of this enzyme in tissue homeostasis and address a potential role in C-to-U RNA editing. Elevated and sustained levels of APOBEC3B led to rapid alteration of cellular fitness, major organ dysfunction, and ultimately lethality in mice. Importantly, extensive analyses of RNA-sequencing and WES from mouse tissues expressing high APOBEC3B levels reveal frequent UCC-to-UUC RNA editing events mainly localized in a specific hotspot. This work identifies, for the first time, a new function for APOBEC3B in RNA editing and presents a valuable preclinical tool to understand the emerging role of APOBEC3B as a potent driver of cancer and other diseases.
Suitable use of FRET-based Biosensors for Quantitative Detection of GPCR Activation
Authors: Brinkenfeldt Nikolaj K. , Dias André , Mathiesen Jesper M. , Martinez Karen L. 
Published: June 2022

AbstractCyclic adenosine 3’,5’-monophosphate is an important second messenger molecule that regulates many downstream signaling pathways in cells. Detection of cAMP levels relies on screenings of cell lysates or the use of genetically encoded biosensors for detection in living cells. Genetically encoded biosensors are, however, primarily used for bioimaging and rarely in high-throughput screenings of potential drug candidates. Here, we describe a quantitative fluorescence-based imaging method based on measurements of single living cells. We used a genetically encoded Epac149 biosensor to investigate cAMP production in living cells following ligand stimulation. The study revealed a dependence of the measured cAMP levels on the expression level of the biosensor in transiently transfected cells. While the biosensor maintained linearity of the signal at high expression levels, the linearity of the biosensor was lost at lower expression levels due to a deficit of the biosensor compared to the maximum possible production of cAMP in the cells. This problem was circumvented by establishment of a stable cell line with constitutive expression of the biosensor. We established dose response curves by stimulation with the β1-adrenergic receptor partial agonist denopamine and observed up to 1.48-fold difference in the cellular response as well as up to 4.27-fold difference in LogEC50 values between cells with insufficient and sufficient biosensor expression. Careful characterization and control of the biosensor expression is therefore important in order to conduct quantitative analysis of the cAMP production and it allows the use of genetically encoded biosensor to be applied in high-throughput screenings.
The dual diagnosis clinician shared care model – a clinical mental health dual diagnosis integrated treatment initiative
Authors: Foster Gavin , Robertson John , Pallis Sophia , Segal Jose 
Published: June 2022

Purpose
To improve outcomes for people with co-occurring mental health and substance use disorders, the Eastern Health Mental Health Service implemented an integrated treatment model known as the dual diagnosis clinician shared care model. This study aims to provide evidence for a relatively unexplored model in clinical mental health services within the state of Victoria, Australia.

Design/methodology/approach
Dual diagnosis clinicians were placed into community mental health clinics in a shared-care, modified case management role, to provide primary and secondary consultations to select consumers and/or their carers, as well as to provide capacity-building training to the mental health staff facing real world clinical challenges in dual diagnosis service delivery.

Findings
Since the commencement of this service, more than 800 consumers of the adult mental health service have been supported to concurrently address their harmful substance use, while receiving recovery-focused community mental health care. Preliminary findings include previously unknown figures on the prevalence for co-occurring substance use at the point of referral and a demonstrated preference by consumers for treatment of both disorders at the same time by the same service (in-house treatment).

Originality/value
The establishment of a dedicated, integrated dual diagnosis team has significantly increased the capacity of a community-based clinical mental health service to engage with and treat consumers with dual diagnosis disorders. This model is beginning to produce evidence challenging traditional siloed approaches to mental health and alcohol and drug treatment.
Multi-objective free-form shape optimization of a synchronous reluctance machine
Authors: Gangl Peter , Köthe Stefan , Mellak Christiane , Cesarano Alessio , Mütze Annette 
Published: June 2022

Purpose
This paper aims to deal with the design optimization of a synchronous reluctance machine to be used in an X-ray tube, where the goal is to maximize the torque while keeping low the amount of material used, by means of gradient-based free-form shape optimization.

Design/methodology/approach
The presented approach is based on the mathematical concept of shape derivatives and allows to obtain new motor designs without the need to introduce a geometric parametrization. This paper presents an extension of a standard gradient-based free-form shape optimization algorithm to the case of multiple objective functions by determining updates, which represent a descent of all involved criteria. Moreover, this paper illustrates a way to obtain an approximate Pareto front.

Findings
The presented method allows to obtain optimal designs of arbitrary, non-parametric shape with very low computational cost. This paper validates the results by comparing them to a parametric geometry optimization in JMAG by means of a stochastic optimization algorithm. While the obtained designs are of similar shape, the computational time used by the gradient-based algorithm is in the order of minutes, compared to several hours taken by the stochastic optimization algorithm.

Originality/value
This paper applies the presented gradient-based multi-objective optimization algorithm in the context of free-form shape optimization using the mathematical concept of shape derivatives. The authors obtain a set of Pareto-optimal designs, each of which is a shape that is not represented by a fixed set of parameters. To the best of the authors’ knowledge, this approach to multi-objective free-form shape optimization is novel in the context of electric machines.
Organizational interventions towards achieving gender equality at the corporate top: a multiple case study approach
Authors: van ‘t Foort-Diepeveen Rosalien A. 
Published: June 2022

PurposeThe aim of this article is twofold: (1) to identify gender equality organizational interventions implemented by a selected number of Dutch companies to increase the number of women at the corporate top and (2) to identify how these interventions overcome barriers to women's advancement and contribute to more women at the corporate top.Design/methodology/approachA comparative case study method was applied through conducting qualitative research. The research was conducted at four large Dutch companies.FindingsThe research identified 23 organizational interventions that were classified in four categories. The cross-case analysis focuses on specific themes, such as the type of interventions, the identified barriers, the successfulness of the interventions and factors contributing to increasing the number of women at the corporate top. The research shows that top-level commitment to this topic is important for the success of interventions and for increasing the number of women at the corporate top and throughout the rest of the organization. Some of the barriers could be overcome by the interventions identified.Practical implicationsThis research provides companies with better insight into the quality and quantity of gender equality organizational interventions implemented by Dutch companies to increase the number of women at the corporate top. It can assist them in deciding which interventions could be implemented in order to achieve gender equality at their corporate top.Originality/valueThe research provides in-depth insight into the types and number of implemented gender equality organizational interventions for women at the corporate top and into the results and perceived effectiveness of such interventions.