bluuebunny/crossref_metadata_2025_split huggingface dataset:
Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability
Authors: Gill Peter , Bleka Øyvind , Fonneløp Ane Elida 
Published: May 2022

AbstractThe application of qPCR to estimate the quantity of DNA present is usually based upon a short amplicon (typically c.80bp) and a longer amplicon (typically c.200-300bp) where the latter is used to determine the amount of degradation present in a sample. The data are used to make decisions about a) whether there is sufficient template to amplify? b) how much of the elution volume to forward to PCR? A typical multiplex amplifies template in the region of 100-500bp. Consequently, the results from an 80bp amplicon will tend to overestimate the actual amplifiable quantity that is present in a degraded sample. To compensate, a method is presented that relates the quantity of amplifiable DNA to the average RFU of the amplified fragments. This provides greatly improved accuracy of the estimated quantity of DNA present, which may differ by more than an order of magnitude compared to qPCR. The relative DNA quantities can be apportioned per contributor once mixture proportions are ascertained with probabilistic genotyping software (EuroForMix). The motivation for this work was to provide an improved method to generate data to prepare distributions that are used to inform activity level propositions. However, other applications will benefit, particularly those where extraction and quantification are bypassed: For example direct PCR and Rapid DNA technology. The overall aim of this work was to provide a method of quantification that is standardised and can be used to compare results between different laboratories that use different multiplexes. A software solution ”ShinyRFU” is provided to aid calculations.
Structural characterization of a breast cancer-associated mutation in caveolin-1
Authors: Han Bing , Gulsevin Alican , Connolly Sarah , Wang Ting , Porta Jason , Tiwari Ajit , Deng Angie , Chang Louise , Peskova Yelena , Mchaoraub Hassane S. , Karakas Erkan , Ohi Melanie D. , Meiler Jens , Kenworthy Anne K. 
Published: May 2022

ABSTRACTCaveolin-1 (CAV1) is a membrane sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a breast cancer-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with wild type CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.
Perceptual decisions exhibit hallmarks of dynamic Bayesian inference
Authors: Charlton Julie A. , Młynarski Wiktor F. , Bai Yoon H. , Hermundstad Ann M. , Goris Robbe L. T. 
Published: May 2022

ABSTRACTTo interpret the sensory environment, the brain combines ambiguous sensory measurements with context-specific prior experience. But environmental contexts can change abruptly and unpredictably, resulting in uncertainty about the current context. Here we address two questions: how should context-specific prior knowledge optimally guide the interpretation of sensory stimuli in changing environments, and do human decision-making strategies resemble this optimum? We probe these questions with a task in which subjects report the orientation of ambiguous visual stimuli that were drawn from three dynamically switching distributions, representing different environmental contexts. We derive predictions for an ideal Bayesian observer that leverages the statistical structure of the task to maximize decision accuracy and show that its decisions are biased by task context. The magnitude of this decision bias is not a fixed property of the sensory measurement but depends on the observer’s belief about the current context. The model therefore predicts that decision bias will grow with the reliability of the context cue, the stability of the environment, and with the number of trials since the last context switch. Analysis of human choice data validates all three predictions, providing evidence that the brain continuously updates probabilistic representations of the environment to best interpret an uncertain, ever-changing world.SIGNIFICANCEThe brain relies on prior knowledge to make perceptual inferences when sensory information is ambiguous. However, when the environmental context changes, the appropriate prior knowledge often changes with it. Here, we develop a Bayesian observer model to investigate how to make optimal perceptual inferences when sensory information and environmental context are both uncertain. The behavioral signature of this strategy is a context-appropriate decision bias whose strength grows with the reliability of the context cue, the stability of the environment, and with the number of decisions since the most recent change in context. We identified exactly this pattern in the behavior of human subjects performing a dynamic orientation discrimination task. Together, our results suggest that the brain continuously updates probabilistic representations of the environment to make perceptual decisions in the face of uncertainty over both sensory and contextual information.
Multiplexed microfluidic platform for stem-cell derived pancreatic islet β cells
Authors: Goswami Ishan , de Klerk Eleonora , Carnese Phichitpol , Hebrok Matthias , Healy Kevin E. 
Published: May 2022

ABSTRACTStem-cell derived β cells offer an alternative to primary islets for biomedical discoveries as well as a potential surrogate for islet transplantation. The expense and challenge of obtaining and maintaining functional stem-cell derived β cells calls for a need to develop better high-content and high-throughput culture systems. Microphysiological systems (MPS) are promising high-content in vitro platforms, but scaling for high-throughput screening and discoveries remain a challenge. Traditionally, simultaneous multiplexing of liquid handling and cell loading poses a challenge in the design of high-throughput MPS. Furthermore, although MPS for islet beta culture/testing have been developed, studies on multi-day culture of stem-cell derived β cells in MPS have been limited. We present a scalable, multiplexed islet beta MPS device that incorporates microfluidic gradient generators to parallelize fluid handling for culture and test conditions. We demonstrated the viability and functionality of the stem-cell derived enriched β clusters (eBCs) for a week, as assessed by the ~2 fold insulin release by the clusters to glucose challenge. To show the scalable multiplexing for drug testing, we demonstrated the exhaustion of eBC insulin reserve after long term exposure to logarithmic concentration range of glybenclamide. The MPS cultured eBCs also revealed a glycolytic bottleneck as inferred by insulin secretion responses to metabolites methyl succinate and glyceric acid. Thus, we present an innovative culture platform for eBCs with a balance of high-content and high-throughput characteristics.
PKN2 deficiency leads both to prenatal ‘congenital’ cardiomyopathy and defective angiotensin II stress responses
Authors: Marshall Jacqueline J T , Cull Joshua J , Alharbi Hajed O , Thin May Zaw , Cooper Susanna TE , Barrington Christopher , Vanyai Hannah , Snoeks Thomas , Siow Bernard , Suáarez-Bonnet Alejandro , Herbert Eleanor , Stuckey Daniel J , Cameron Angus , Prin Fabrice , Cook Andrew C. , Priestnall Simon L , Chotani Sonia , Rackham Owen J L , Meijles Daniel N , Mohun Tim , Clerk Angela , Parker Peter J 
Published: May 2022

ABSTRACTBackgroundThe protein kinase PKN2 is required for embryonic development, and PKN2 knockout mice die as a result of failure in expansion of mesoderm tissues, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established.MethodsWe used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency. Cardiac function and dimensions were assessed with high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were assessed.ResultsCardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed upregulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, this was explored using mice with constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis.ConclusionsCardiomyocyte PKN2 is essential for heart development and formation of compact myocardium, and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.
Cyclin A and Cks1 promote kinase consensus switching to non-proline directed CDK1 phosphorylation
Authors: al-Rawi Aymen , Korolchuk Svitlana , Endicott Jane , Ly Tony 
Published: May 2022

SummaryOrdered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation. Increases in CDK1 activity alters substrate choice, with intermediate and low sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence was shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 promote non-proline directed phosphorylation, preferably on sites with a +3 lysine residue. Indeed, 70% of cell cycle regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline directed CDK1 sites.
Phase-separated nuclear bodies of nucleoporin fusions, SET-NUP214 and NUP98-HOXA9, promote condensation of MLL1 and CRM1 to activate target genes
Authors: Oka Masahiro , Otani Mayumi , Miyamoto Yoichi , Adachi Jun , Tomonaga Takeshi , Asally Munehiro , Ohkawa Yasuyuki , Yoneda Yoshihiro 
Published: May 2022

ABSTRACTNucleoporins NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies. However, the function and physiological significance of these nuclear bodies remain largely unknown. Previously, we reported that both NUP98-HOXA9 and SET-NUP214 are recruited to HOX cluster regions via chromatin-bound CRM1, a nuclear export receptor (Oka et al., 2019). Here, we show that these nuclear bodies promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase which is essential for the maintenance of HOX gene expression. Our analysis revealed that SET-NUP214 and CRM1 robustly associate with MLL1 to form nuclear bodies and are colocalized on chromatin. We also showed that MLL1 and CRM1 are recruited to the nuclear bodies of NUP98-HOXA9 and that the NUP98-HOXA9/CRM1/MLL1 complex accumulates on its target gene loci, including HOX clusters and MEIS1. These phenomena were not observed in phase-separation–deficient mutants or non-DNA-binding mutants of NUP98-HOXA9. Collectively, these results show that both phase separation and proper targeting of nucleoporin fusions to specific sites could enhance the activation of a wide range of target genes by promoting the condensation of MLL1 and CRM1.
The AMPK activator A-769662 inhibits human TASK3 potassium channels in an AMPK-independent manner
Authors: Said Esraa A. , Lewis Ryan W. , Dallas Mark L. , Peers Chris , Ross Fiona A. , Hardie D. Grahame , Evans A. Mark 
Published: May 2022

ABSTRACTHeteromeric TASK1/3 channels play a fundamental role in oxygen-sensing by carotid body type 1 cells, where hypoxia-induced inhibition of TASK3 and/or TASK1/3 potassium currents leads to depolarisation, voltage-gated calcium entry, exocytotic transmitter release and increases in carotid body afferent input responses that initiate corrective changes in breathing patterns. However, the mechanism by which hypoxia leads to TASK-1/3 channel inhibition is still debated. It had been proposed that the AMP-activated protein kinase (AMPK) might directly phosphorylate and inhibit TASK channels, in particular TASK-3, although subsequent studies on rat type I cells argued against this view. Here we report on the effects of novel, highly selective AMPK activators on recombinant human TASK-3 potassium channels. Sequence alignment identified an AMPK recognition motif in TASK-3, but not TASK-1, with Ser55 representing a potential site for AMPK-dependent phosphorylation in TASK-3. However, neither of the AMPK activators, AICAR or MK-8722, caused a significant reduction of human TASK-3 current amplitude. By contrast, high concentrations of the AMPK activator A-769662 (100-500 µM) inhibited human TASK-3 currents in a concentration-dependent manner. Importantly, A-769662 (300 µM) also inhibited human TASK-3 channels in HEK293 cells that stably over-expressed an AMPK-β1 subunit mutant (S108A) that renders AMPK insensitive to activators binding the Allosteric Drug and Metabolite (ADaM) site, such as A-769662. We therefore identify A-769662 as a novel human TASK-3 channel inhibitor and provide conclusive evidence that AMPK does not regulate TASK-3 channel currents.
Morphological profiling by high-throughput single-cell biophysical fractometry
Authors: Zhang Ziqi , Lee Kelvin C. M. , Siu Dickson M. D. , Lai Queenie T. K. , Lam Edmund Y. , Tsia Kevin K. 
Published: May 2022

AbstractComplex and irregular cell architecture is known to statistically exhibit fractal geometry, i.e., a pattern resembles a smaller part of itself. Although fractal variations in cells are proven to be closely associated with the disease-related phenotypes that are otherwise obscured in the standard cell-based assays, fractal analysis with single-cell precision remains largely unexplored. To close this gap, here we develop an image-based approach that quantifies a multitude of single-cell biophysical fractal-related properties at subcellular resolution. Taking together with its high-throughput single-cell imaging performance (~10,000 cells/sec), this technique, termed single-cell biophysical fractometry, offers sufficient statistical power for delineating the cellular heterogeneity, in the context of classification of lung-cancer cell subtypes and tracking of cell-cycle progression. Further correlative fractal analysis shows that single-cell biophysical fractometry can enrich the standard morphological profiling depth and spearhead systematic fractal analysis of how cell morphology encodes cellular health and pathological conditions.
Sickle red blood cell derived extracellular vesicles activate endothelial cells and enhance sickle red cell adhesion mediated by von Willebrand factor
Authors: An Ran , Man Yuncheng , Cheng Kevin , Zhang Tianyi , Chen Chunsheng , Kucukal Erdem , Wulftange William J. , Goreke Utku , Bode Allison , Nayak Lalitha V. , Vercellotti Gregory M. , Belcher John D. , Little Jane A. , Gurkan Umut A. 
Published: May 2022

ABSTRACTEndothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles, REVs, are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signaling and transcriptional regulation at longer terms. However, the SS REV-mediated short term non transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 hours. Compared with AA REVs, SS REVs promoted human pulmonary microvascular endothelial cells (HPMEC) activation indicated by increased von Willebrand Factor (vWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by vWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, studies in SS mice with implanted dorsal skin-fold chambers found hemin-induced stasis was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable, and was higher with SS RBCs from patients with increased markers of hemolysis (LDH and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasize the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using vWF as a potential target.
An enhanced sensorless control based on active disturbance rejection controller for a PMSM system: design and hardware implementation
Authors: Lu Hao , Li Shengquan , Feng Bo , Li Juan 
Published: May 2022

Purpose
This paper mainly aims to deal with the problems of uncertainties including modelling errors, unknown dynamics and disturbances caused by load mutation in control of permanent magnet synchronous motor (PMSM).

Design/methodology/approach
This paper proposes an enhanced speed sensorless vector control method based on an active disturbance rejection controller (ADRC) for a PMSM. First, a state space model of the PMSM is obtained for the field orientation control strategy. Second, a sliding mode observer (SMO) based on back electromotive force (EMF) is introduced to replace the encode to estimate the rotor flux position angle and speed. Third, an infinite impulse response (IIR) filter is introduced to eliminate high frequency noise mixed in the output of the sliding mode observer. In addition, a speed control method based on an extended state observer (ESO) is proposed to estimate and compensate for the total disturbances. Finally, an experimental set-up is built to verify the effectiveness and superiority of the proposed ADRC-based control method.

Findings
The comparative experimental results show that the proposed speed sensorless control method with the IIR filter can achieve excellent robustness and speed tracking performance for PMSM system.

Research limitations/implications
An enhanced sensorless control method based on active disturbance rejection controller is designed to realize high precision control of the PMSM; the IIR filter is used to attenuate the chattering problem of traditional SMO; this method simplifies the system and saves the total cost due to the speed sensorless technology.

Practical implications
The use of sensorless can reduce costs and be more beneficial to actual industrial application.

Originality/value
The proposed enhanced speed sensorless vector control method based on an ADRC with the IIR filter enriches the control method of PMSM. It can ameliorate system robustness and achieve excellent speed tracking performance.
Consumer purchase intention of intangible cultural heritage products (ICHP): effects of cultural identity, consumer knowledge and manufacture type
Authors: Zhang Mengxia , Guo Xixuan , Guo Xiaoling , Jolibert Alain 
Published: May 2022

PurposeIntangible cultural heritage products (ICHP) in this paper refer to the products made with handicrafts listed as intangible cultural heritage. The purpose of this paper is to examine the effects of consumer cultural identity and consumer knowledge on purchase intentions of ICHP, as well as the role of the perceived scarcity for such effects.Design/methodology/approachThree between-participants experiments were conducted to investigate the effect of cultural identity on purchase intention of ICHP and the underlying mechanism of the effect.FindingsThe results show that cultural identity has a positive influence on ICHP purchase intention, and this effect is stronger for consumers with higher level of consumer knowledge. Furthermore, perceived scarcity underlies such effect, but the scarcity account holds only for handmade ICHP, not for machine-manufactured products.Practical implicationsThe current research suggests that ICHP should remain handmade to preserve their scarcity as a distinctive feature. Additionally, business practitioners handling ICHP should target consumers of high cultural identity, and/or provide iconic cues to activate their cultural identity situationally. Furthermore, it highlights the importance of consumer education programs about ICHP-related knowledge in enhancing the cultural identity effect.Originality/valueThis paper explores systematically the conditions and process of consumers' responses toward ICHP for the first time. Besides, it builds on accessibility-diagnosticity framework and provides novel knowledge about the functioning of consumer cultural identity. It also enriches our understanding of perceived scarcity from the supply side.