bluuebunny/crossref_metadata_2025 huggingface dataset:
kcna1a mutant zebrafish as a model of episodic ataxia type 1 and epilepsy
Authors: Dogra Deepika , Meza-Santoscoy Paola L. , Rehak Renata , de la Hoz Cristiane L R , Gavrilovici Cezar , Ibhazehiebo Kingsley , Rho Jong M. , Kurrasch Deborah M.
Published: September 2022

Objective

KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), with epilepsy as a common comorbidity. Current medications only provide partial relief to ataxia and/or seizures, making new drugs needed. Here, we investigate the utility of zebrafish kcna1a−/− as a model of EA1 with epilepsy by characterizing its phenotype and comparing the efficacy of the first-line therapy carbamazepine in kcna1a−/− zebrafish to Kcna1−/− rodents.

Methods

We used CRISPR/Cas9 mutagenesis to introduce a mutation in the sixth segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a−/− larvae to assess ataxia- and epilepsy-related phenotypes. We also carried out real-time qPCRs to measure the transcript levels of brain hyperexcitability markers and bioenergetic profiling of kcna1a−/− larvae to evaluate their metabolic health. Carbamazepine efficacy was tested using behavioral assessments in kcna1a−/− zebrafish and seizure frequency in Kcna1−/− mice.

Results

kcna1a−/− zebrafish showed uncoordinated movements and locomotor deficits. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation. Extracellular field recordings and upregulated fosab transcript levels showed hyperexcitability of the kcna1a−/− brain. Further, vglut2a and gad1b transcript levels were altered, indicative of neuronal excitatory/inhibitory imbalance in the kcna1a−/− brain. Metabolic health was also compromised in kcna1a−/− as seen by a significant reduction in measures of cellular respiration. Notably, carbamazepine reduced the impaired startle response in kcna1a−/− zebrafish but had no effect on the seizure frequency in Kcna1−/− mice, suggesting that this EA1 zebrafish model might better translate to human efficacy compared to rodents.

Significance

We conclude that zebrafish kcna1a−/− larvae show ataxia and epilepsy-related phenotypes and that they are responsive to carbamazepine treatment, consistent with EA1 patients. This study supports the notion that these zebrafish disease models can be useful for drug screening as well as studying the underlying disease biology.

KEY POINTS

Zebrafish kcna1a−/− larvae display dynamic behavioral changes, along with ataxia-like uncoordinated movements and brain hyperexcitability

kcna1a−/− larvae have dysfunctional neuronal excitatory/inhibitory balance and perturbed metabolic health

Similar to its effectiveness in patients, carbamazepine treatment improves behavioral deficits in kcna1a−/− larvae
Macaque amygdala, claustrum and pulvinar support the cross-modal association of social audio-visual stimuli based on meaning
Authors: Froesel Mathilda , Gacoin Maëva , Clavagnier Simon , Hauser Marc , Goudard Quentin , Ben Hamed Suliann
Published: September 2022

Social communication draws on several cognitive functions such as perception, emotion recognition and attention. In a previous study, we demonstrated that macaques associate audiovisual information when processing their species-specific communicative signals. Specifically, cortical activation is inhibited when there is a mismatch between vocalisations and social visual information whereas activation is enhanced in the lateral sulcus, superior temporal sulcus as well as a larger network composed of early visual and prefrontal areas when vocalisations and social visual information match. Here, we use a similar task and functional magnetic resonance imaging to assess the role of subcortical structures. We identify three subcortical regions involved in audio-visual processing of species-specific communicative signal: the amygdala, the claustrum and the pulvinar. Like the cortex, these subcortical structures are not activated when there is a mismatch between visual and acoustic information. In contrast, the amygdala and claustrum are activated by visual, auditory congruent and audio-visual stimulations. The pulvinar responds in a task-dependent manner, along a specific spatial sensory gradient. Anterior pulvinar responds to auditory stimuli, medial pulvinar is activated by auditory, audio-visual and visual stimuli and the dorsal lateral pulvinar only responds to visual stimuli in a pure visual task. The medial pulvinar and the amygdala are the only subcortical structures integrating audio-visual social stimuli. We propose that these three structures belong to a multisensory network that modulates the perception of visual socioemotional information and vocalizations as a function of the relevance of the stimuli in the social context.

Significance Statement

Understanding and correctly associating socioemotional information across sensory modalities, such that happy faces predict laughter and escape scenes screams, is essential when living in complex social groups. Using functional magnetic imaging in the awake macaque, we identify three subcortical structures – amygdala, claustrum and pulvinar - that only respond to auditory information that matches the ongoing visual socioemotional context, such as hearing positively valenced coo calls and seeing positively valenced grooming monkeys. We additionally describe task-dependent activations in the pulvinar, organizing along a specific spatial sensory gradient, supporting its role as a network regulator.
Fluorescence lifetime enables high-resolution analysis of neuromodulator dynamics across time and animals
Authors: Ma Pingchuan , Chen Peter , Tilden Elizabeth , Aggarwal Samarth , Oldenborg Anna , Chen Yao
Published: September 2022

The dynamics of neuromodulators are essential for their functions. Optical sensors have transformed the study of neuromodulators because they capture neuromodulator dynamics with high spatial and temporal resolution. However, fluorescence intensity-based sensors are restricted to measure acute changes within one animal over a short period because intensity varies with sensor expression level and excitation light fluctuation. In contrast, fluorescence lifetime is impervious to sensor expression level or excitation light power, allowing comparison between individuals and across long periods. Here, we discover fluorescence lifetime response in multiple intensity-based neuromodulator sensors. Using the acetylcholine sensor GRABACh3.0to investigate the power of lifetime measurement, we find that fluorescence lifetime correlates with animal behavior states accurately despite varying excitation power or changes in sensor expression level across weeks and animals. Thus, fluorescence lifetime of neuromodulator sensors enables comparison of neuromodulator dynamics at high resolution between animals and for chronic time scales.
A simple non-invasive C reactive protein-based score can predict outcome in patients with COVID-19
Authors: Scotto Riccardo , Lanzardo Amedeo , Buonomo Antonio Riccardo , Pinchera Biagio , Letizia Cattaneo , Sardanelli Alessia , Mercinelli Simona , Viceconte Giulio , Zappulo Emanuela , Villari Riccardo , Foggia Maria , Gentile Ivan ,   Federico II COVID-team
Published: September 2022

Background

We evaluated the role of CRP and other laboratory parameters in predicting the worsening of clinical conditions during hospitalization, ICU admission and fatal outcome among patients with COVID-19.

Methods

We enrolled consecutive adult inpatients with SARS-CoV-2 infection and respiratory symptoms treated in three different COVID centres. We looked for laboratory parameters collected within 48 hours from hospital admission as predictors of clinical condition.

Results

Three-hundred ninety patients were included in the study. At the correlation and regression analysis, age, baseline CRP and LDH were associated with a P/F ratio<200 during hospitalization. At the multivariate analysis, male gender and CRP > 60 mg/l at admission showed to be independently associated with ICU admission. Lymphocytes<1000 cell/μL at admission were associated with worst P/F ratio. The only laboratory predictor of fatal outcome was CRP>60 mg/l at admission. Based on these results, we devised an 11-points numeric ordinary score based on age, sex, CRP and LDH at admission (ASCL score). Patients with ASCL score of 0 or 2 showed to be protected against a P/F ratio<200, while patients with ASCL score of 6, 7 and 8 showed to be at risk for P/F ratio<200. Patients with ASCL score≥7 had a significant increase to die during the hospitalization.

Conclusions

Patients with CRP>60 mg/l or LDH>300 IU/l at hospital admission, as well as patients with an ASCL score>6 at hospital admission, should be prioritized for careful respiratory function monitoring and early treatment to prevent a progression of the disease.
Production of selenium nanoparticles occurs through an interconnected pathway of sulfur metabolism and oxidative stress response inPseudomonas putidaKT2440
Authors: Avendaño Roberto , Muñoz-Montero Said , Rojas-Gätjens Diego , Fuentes Paola , Vieto Sofía , Montenegro Rafael , Salvador Manuel , Frew Rufus , Kim Juhyun , Chavarría Max , Jiménez Jose I.
Published: September 2022

The soil bacteriumPseudomonas putidaKT2440 has been shown to produce selenium nanoparticles aerobically from selenite; however, the molecular actors involved in this process are unknown. Here, through a combination of genetic and analytical techniques, we report the first insights into selenite metabolism in this bacterium. Our results suggest that the reduction of selenite occurs through an interconnected metabolic network involving central metabolic reactions, sulfur metabolism, and the response to oxidative stress. Genes such assucA, D2HGDH and PP_3148 revealed that the 2-ketoglutarate and glutamate metabolism is important to converting selenite into selenium. On the other hand, mutants affecting the activity of sulfite reductase reduced the bacteria’s ability to transform selenite. Other genes related to sulfur metabolism (ssuEF,sfnCE, sqrR,sqrandpdo2) and stress response (gqr,lsfA,ahpCFandsadI) were also identified as involved in selenite transformations. Interestingly, suppression of genessqrR,sqrandpdo2resulted in the production of selenium nanoparticles at a higher rate than the wild-type strain, which is of biotechnological interest. The data provided in this study brings us closer to understanding the metabolism of selenium in bacteria, and offers new targets for the development of biotechnological tools for the production of selenium nanoparticles.
Location of CD39+ T cell sub-populations within tumours predict differential outcomes in non-small cell lung cancer
Authors: Koppensteiner Lilian , Mathieson Layla , Pattle Samuel , Dorward David A , O’Connor Richard , Akram Ahsan
Published: September 2022

An improved mechanistic understanding of immunosuppressive pathways in NSCLC is important to develop novel diagnostic and therapeutic approaches. Here, we reveal that the prognostic significance of the rate limiting ectonucleotidases in adenosine production CD39 and CD73 requires knowledge of cell type specific expression and localisation within tumours. In a cohort of early treatment naïve NSCLC patients, high stromal expression of CD39 and CD73 predicts poor outcome. CD39 expression amongst T cells identifies CD39+CD4+ Tregs which predict poor outcome and CD39+CD103+ CTL which confer a survival benefit if high densities are observed inside of the tumour nest. Bulk RNA Seq shows that the TME of NSCLC upregulates regulatory pathways in CD4+ T cells and exhaustion in CD8+ T cells. Analysis of single-cell RNASeq datasets illustrates that CD39+CD4+ Tregs are enriched in Treg signature gene sets, and CD39+CD103+ CTL show gene signatures indicative of an exhausted cytotoxic phenotype with an upregulated expression of CXCL13.

Combined knowledge of patterns of distribution and location are required to understand the prognostic impact of CD39+ T cell populations in NSCLC. This study provides an improved understanding of the spatial and functional characteristics of CD39+ T cells and illustrates their significance to patient outcome.
Lineage segregation in human pre-implantation embryos is specified by YAP1 and TEAD1
Authors: Regin Marius , Essahib Wafaa , Demtschenko Andrej , Dewandre Delphine , David Laurent , Gerri Claudia , Niakan Kathy , Verheyen Greta , Tournaye Herman , Sterckx Johan , Sermon Karen , Van de Velde Hilde
Published: September 2022

We know that polarity and YAP1 play a key role in trophectoderm initiation in compacted human embryos, however we know little about the TEAD family of transcription factors that become activated by YAP1 and especially if they play a role during epiblast and primitive endoderm formation. Here we show that compaction occurs heterogeneously between the 8- and 16-cell stages. While 8-cell stage blastomeres are not yet polarized, polarized outer cells and non-polarized inner cells arise in compacted 16-cell stage embryos. While trophectoderm specifiers TEAD1, YAP1 and GATA3 mostly co-localise in the nuclei of polarized outer/trophectoderm cells, they are also found in some cells of compacting embryos before polarity is established indicating that differentiation into trophectoderm cells can be initiated independently of polarity. In the inner cell mass, TEAD1 and YAP1 also distinguish GATA4 positive cells in a salt-and-pepper distribution and in the sorted primitive endoderm cells. Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the road for further functional studies. Fundamental knowledge of lineage segregation events will eventually explain how and why embryos fail to develop further before or during implantation.
Including both sexes in in vivo research does not necessitate an increase in sample size: a key role for factorial analysis methods
Authors: Phillips Benjamin , Haschler Timo N. , Karp Natasha A.
Published: September 2022

In recent years, there has been a strong drive to improve the inclusion of animals of both sex during in vivo research, driven by a need to improve sex representation in fundamental biology and drug development. This has resulted in inclusion mandates by funding bodies and journals, alongside numerous published manuscripts highlighting the issue and providing guidance to scientists. However, progress is slow and blockers to the routine use of both sexes remain. From a statistical and experimental design perspective, concerns include difficulty selecting and conducting an appropriate analysis and the perceived need for a higher sample size to achieve an equivalent level of statistical power. When both sexes are included, analysis errors are frequent, including inappropriate pooling or sex-disaggregation of the data. These mistakes result in a failure to properly account for the variation in the data that arises from sex, and subsequently lead to poor inference regarding the biological impact of sex. The purpose of this manuscript is to address frequently cited blockers and analysis errors, thus providing a practical guide to support scientists in the design of in vivo studies which include both sexes. Primarily, we demonstrate that there is no loss of power to detect treatment effects when splitting the sample size across sexes in most common biological scenarios, providing that the data are analysed appropriately. In the rare situations where power is lost, the benefit of understanding the role of sex outweighs the power considerations. When estimating a generalisable translatable effect, where exploring sex differences are not the primary scientific objective, we recommend splitting the sample size across male and female mice as a standard strategy. We also demonstrate an optimal analysis pipeline for analysing data gathered using both sexes which is designed to help address common analysis errors.
MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control
Authors: Heiland Mona , Connolly Niamh M. C. , Nguyen Ngoc T. , Kesavan Jaideep C. , Fanning Kevin , Sanfeliu Albert , Yan Yan , Venø Morten T. , Costard Lara S. , Neubert Valentin , Hill Thomas D. M. , Rosenow Felix , Bauer Sebastian , Kjems Jørgen , Morris Gareth , Henshall David C.
Published: September 2022

There remains an urgent need for new therapies for drug-resistant epilepsy (DRE). Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bi-directional control of sodium channel expression are needed. MicroRNAs (miRNA) are small non-coding RNAs which negatively regulate gene expression. Here, we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the novel anti-seizure medicine cannabidiol (CBD) and plasma from patients with DRE, converge on a single target, miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking of miR-335-5p also increased voltage-gated sodium currents in human iPSC-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazole seizure model, whereas AAV9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect brain excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide new biomarker or therapeutic strategies for different types of drug-resistant epilepsy.

Significance Statement

Despite the clinical availability of over 30 anti-seizure medications (ASMs), around 30% of people with epilepsy do not achieve seizure freedom. MicroRNAs are small non-coding RNAs which negatively regulate protein expression by binding to target mRNAs. Here, we identified the brain-enriched miR-335-5p to be commonly altered in three heterogenous miRNA profiling datasets. Bi-directional modulation of miR-335-5p identified a potential homeostatic role of miR-335-5p in brain excitability involving voltage-gated sodium channels. Electrophysiological and in vivo approaches revealed pro-epileptic activity of miR-335-5p inhibition whereas overexpression of miR-335-5p resulted in anti-epileptic activity. Overall, targeting miR-335-5p could provide a new approach in the modulation of brain excitability, with possible therapeutic applications in drug-resistant epilepsies and other neurological diseases.
Structural and functional characterization of chloroplast ribulose-5-phosphate-3-epimerase from the model green microalgaChlamydomonas reinhardtii
Authors: Meloni Maria , Fanti Silvia , Tedesco Daniele , Gurrieri Libero , Trost Paolo , Fermani Simona , Lemaire Stéphane D. , Zaffagnini Mirko , Henri Julien
Published: September 2022

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 fromChlamydomonas reinhardtii(CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of anα8β8triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.
Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from larval breeding sites
Authors: Kristan Mojca , Acford-Palmer Holly , Campos Monica Oliveira , Collins Emma , Phelan Jody , Portwood Natalie M. , Pelloquin Bethanie , Clarke Sian , Lines Jo , Clark Taane G. , Walker Thomas , Campino Susana , Messenger Louisa A.
Published: September 2022

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 32 second instar larvae in 50ml of water. An. stephensi eDNA, derived from emergent pupae for 24 hours, was remarkably stable, and still detectable ~2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.
LKB1 regulates JNK-dependent stress signaling and apoptotic dependency ofKRAS-mutant lung cancers
Authors: Li Chendi , Syed Mohammed Usman , Nimbalkar Anahita , Shen Yi , Fraser Cameron , Inde Zintis , Qin Xingping , Ouyang Jian , Kreuzer Johannes , Clark Sarah E. , Kelley Grace , Hensley Emily , Morris Robert , Lazaro Raul , Belmontes Brian , Oh Audris , Walcott Makeba , Nabel Christopher , Caenepeel Sean , Saiki Anne Y. , Rex Karen , Lipford J. Russell , Heist Rebecca S. , Lin Jessica J. , Haas Wilhelm , Sarosiek Kristopher , Hughes Paul E. , Hata Aaron N.
Published: September 2022

The efficacy of molecularly targeted anti-cancer therapies may be limited by the presence of co-occurring mutations within a tumor1–3. Conversely, these alterations may confer collateral vulnerabilities that can be leveraged for the development of novel therapeutic approaches.KRAS-mutant lung cancers are distinguished by recurrent inactivating mutations in the tumor suppressorSTK11/LKB14that facilitate tumorigenesis by modulating energy balance5, 6, enhancing metastatic potential7,8and enabling immune evasion9,10. However, whether LKB1 plays a role in modulating cellular responses to therapeutic stress is largely unknown. Here we show that LKB1 suppresses JNK-dependent stress signaling inKRAS-mutant lung cancer cells upon acute loss of oncogenic signaling. In LKB1-deficientKRAS-mutant cells, inhibition of KRAS or its downstream effector MEK leads to hyperactivation of JNK due to loss of NUAK-mediated PP1B phosphatase activity. JNK-mediated inhibitory phosphorylation of BCL-XL rewires apoptotic dependencies, rendering LKB1-deficient cells vulnerable to MCL-1 inhibition. These results uncover a previously unknown role for LKB1 in regulating stress signaling and the mitochondrial apoptotic response of cancer cells independent of its tumor suppressor activity mediated by AMPK11–13and SIK14,15kinases. Additionally, our study reveals a therapy-induced vulnerability in LKB1-deficientKRAS-mutant lung cancer cells that could be exploited as a genotype-informed strategy to improve the efficacy of KRAS-targeted therapies.
Modeling Microtubule-Cytoplasm Interaction in Plant Cells
Authors: Murshed Mohammad , Wei Donghui , Gu Ying , Wang Jin
Published: September 2022

Although microtubules in plant cells have been extensively studied, the mechanisms that regulate the spatial organization of microtubules are poorly understood. We hypothesize that the interaction between microtubules and cytoplasmic flow plays an important role in the assembly and orientation of microtubules. To test this hypothesis, we developed a new computational modeling framework for microtubules based on theory and methods from the fluid-structure interaction. We employed the immersed boundary method to track the movement of microtubules in cytoplasmic flow. We also incorporated details of the encounter dynamics when two microtubules collide with each other. We verified our computational model through several numerical tests before applying it to the simulation of the microtubule-cytoplasm interaction in a growing plant cell. Our computational investigation demonstrated that microtubules are primarily oriented in the direction orthogonal to the axis of cell elongation. We validated the simulation results through a comparison with the measurement from laboratory experiments. We found that our computational model, with further calibration, was capable of generating microtubule orientation patterns that were qualitatively and quantitatively consistent with the experimental results. The computational model proposed in this study can be naturally extended to many other cellular systems that involve the interaction between microstructures and the intracellular fluid.
A NOVEL UNBIASED SEED-BASED RNAi SCREEN IDENTIFIES SMALL RNAs THAT INHIBIT ANDROGEN SIGNALING AND PROSTATE CANCER CELL GROWTH
Authors: Corbin Joshua M , Georgescu Constantin , Wang Lin , Wren Jonathan D , Bieniasz Magdalena , Xu Chao , Asch Adam S , Ruiz-Echevarría Maria J
Published: September 2022

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here we explore a therapeutic strategy for PCa based on the ability of sh/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3’-UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell cycle genes. When tested in mice, the toxic shRNAs also inhibited castration resistant PCa and exhibited therapeutic efficacy in pre-established tumors. This multi-targeted RNAi approach may be a promising therapeutic strategy for PCa.
Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing during epigenetic reprogramming in pollen
Authors: Ichino Lucia , Picard Colette L. , Yun Jaewon , Chotai Meera , Wang Shuya , Lin Evan Kai , Papareddy Ranjith K. , Xue Yan , Jacobsen Steven E.
Published: September 2022

Silencing of transposable elements (TEs) drove the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here we show via single-nucleus RNA-seq of developing male gametophytes that these repressors are critical for TE silencing in the pollen vegetative cell, which undergoes epigenetic reprogramming causing chromatin decompaction to support fertilization by sperm cells. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We found that TEs repressed by MBD5/6 gain accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system especially important when chromatin compaction is compromised.
Structural basis of Norrin recognition by Lgr4/5/6
Authors: Chang Tao-Hsin , Harlos Karl , Hsieh Fu-Lien , Jones E. Yvonne
Published: September 2022

Lgr4/5/6 have been identified as stem cell markers and canonical Wnt signalling enhancers via complexing with R-spondin and Rnf43/Znrf3. Lgr4/5/6 have been reported to interact with Norrin, a cystine-knot growth factor that activates Wnt signalling via binding to Frizzled4 cysteine-rich domain (Fz4CRD). Norrin–Fz4 signalling axis regulates central nervous system vascularization. However, the molecular basis of Norrin and Lgr4/5/6 recognition remains lacking. Here, we present the structure of Norrin in complex with an ectodomain variant of Lgr4 (Norrin–Lgr4ECD), revealing a 2:2 stoichiometry. Our analyses show that residues of Lgr4/5/6ECD for Norrin binding are highly conserved. Structural comparisons of Norrin–Lgr4ECD with Rspo1– Lgr4ECD and Norrin–Fz4CRD complexes reveal that Norrin binding site on Lgr4 overlaps with Rspo1 binding site on Lgr4 and Fz4 binding site on Norrin overlaps with Lgr4 binding site on Norrin. The present work opens new avenues to elucidate the function of Norrin–Lgr4/5/6 signalling axis.
Stress vesicles are induced by acute mechanical force and precede the commitment of epidermal stem cells to terminal differentiation
Authors: Huang Sixia , Kuri Paola , Zou Jonathan , Blanco Adriana , Marshall Maxwell , Rice Gabriella , Prouty Stephen , Dentchev Tzvete , Doepner Miriam , Boerckel Joel D. , Capell Brian C. , Ridky Todd W. , Rompolas Panteleimon
Published: September 2022

The skin has a pronounced ability to adapt to physical changes in the environment by exhibiting plasticity at the cellular level. Transient mechanical deformations applied to the skin are accommodated without permanent changes to tissue structure. However, sustained physical stress induces long-lasting alterations in the skin, which are mediated by shifts in the fates of epidermal stem cells. To investigate this phenomenon, we implemented two-photon intravital imaging to capture the responses of epidermal cells when an acute mechanical force is applied to the live skin. We show that mechanical stress induces the formation of intracellular vesicles in epidermal stem cells, which are filled with extracellular fluid and gradually enlarge, causing the deformation of the cell nucleus. By lineage tracing analysis we demonstrate that the degree of nuclear deformation is linked to cell fate. Utilizing a fluorescentin vivoreporter, to capture intracellular calcium dynamics, we show that mechanical force induces a sustained increase in intracellular calcium within basal epidermal stem cells. Conditional deletion of Piezo1, a mechanosensitive ion channel, alters intracellular calcium dynamics and increases the number of stress vesicles in epidermal stem cells. Using a human skin xenograft model, we show that stress vesicles are a conserved phenomenon in mammalian skin. This study uncovers stress vesicles as key manifestations of the mechanism that regulates the fate of epidermal stem cells under conditions of mechanical stress, in which Piezo1 and calcium dynamics are also involved.
Detyrosination enrichment on microtubule subsets is established by the interplay between a stochastically-acting enzyme and microtubule stability
Authors: Tang Qing , Sensale Sebastian , Bond Charles , Qiao Andy , Hugelier Siewert , Arab Arian , Arya Gaurav , Lakadamyali Melike
Published: September 2022

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions ranging from intracellular transport to chromosome segregation. Yet, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, which is the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that, in certain cell types, only a small subpopulation of microtubules is highly enriched with the detyrosination mark (∼30%) and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, Vasohibin (VASH) leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated α-tubulin subunits after Nocodazole washout. Microtubule detyrosination accumulates slowly and in a disperse pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short time scale suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we propose a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.
in vivo RNA structural probing of guanine and uracil nucleotides in yeast
Authors: Xiao Kevin , Ghalei Homa , Khoshnevis Sohail
Published: September 2022

RNAs have critical catalytic or regulatory functions in the cell and play significant roles in many steps of gene expression regulation. RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structural probing is an indirect method to probe the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. The chemical modifications can target either the RNA backbone or the Watson-Crick face of nucleotides. The selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) can probe the ribose sugar in all unpaired RNA nucleotides. In contrast, Dimethyl sulfate (DMS) alkylates adenine and cytosine and reports on base pairing context but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in the plant model system Oryza sativa and in the prokaryotic organisms Bacillus subtilis and Echerichia coli, as well as human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyzed the effectiveness of guanine modification by a family of aldehyde derivatives, the glyoxal family, in Saccharomyces cerevisiae and Candida albicans. We also explored the effectiveness of uracil modification by carbodiimide N-cyclohexyl-N-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) in vivo. We show that among the glyoxal family, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. We also demonstrate uracil modification by CMCT in S. cerevisiae in vivo. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.
In situtargeted mutagenesis of gut bacteria
Authors: Brödel Andreas K , Charpenay Loïc , Galtier Matthieu , Fuche Fabien J , Terrasse Rémi , Poquet Chloé , Arraou Marion , Prevot Gautier , Spadoni Dalila , Hessel Edith M , Fernandez-Rodriguez Jesus , Duportet Xavier , Bikard David
Published: September 2022

Microbiome research is revealing a growing number of bacterial genes that impact our health. While CRISPR-derived tools have shown great success in editing disease-driving genes in human cells, we currently lack the tools to achieve comparable success for bacterial targets. Here we engineer a phage-derived particle to deliver a base editor and modifyE. colicolonizing the mouse gut. This was achieved using a non-replicative DNA payload, preventing maintenance and dissemination of the payload, while allowing for an editing efficiency of up to 99.7% of the target bacterial population. The editing of a β-lactamase gene resulted in the stable maintenance of edited bacteria in the mouse gut at least 42 days after treatment. By enabling thein situmodification of bacteria directly in the gut, our approach offers a novel avenue to investigate the function of bacterial genes and provides an opportunity to develop novel microbiome-targeted therapies.
Single-particle tracking reveals heterogeneous PIEZO1 diffusion
Authors: Ly Alan T. , Freites J. Alfredo , Bertaccini Gabriella A. , Evans Elizabeth L. , Dickinson George D. , Tobias Douglas J. , Pathak Medha M.
Published: September 2022

The mechanically-activated ion channel PIEZO1 is critical to numerous physiological processes, and is activated by diverse mechanical cues. The channel is gated by membrane tension and has been found to be mobile in the plasma membrane. We employed single particle tracking (SPT) of endogenous, tdTomato-tagged PIEZO1 using Total Internal Reflection Fluorescence Microscopy in live cells. Application of SPT unveiled a surprising heterogeneity of diffusing PIEZO1 subpopulations, which we labeled “mobile” and “immobile”. We sorted these trajectories into the two aforementioned categories using trajectory spread. To evaluate the effects of the plasma membrane composition on PIEZO1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol, or by adding margaric acid to stiffen the membrane. To examine effects of channel activation on PIEZO1 mobility, we treated cells with Yoda1, a PIEZO1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that cholesterol removal and Yoda1 incubation increased the channel’s propensity for mobility. Conversely, we found that GsMTx-4 incubation and cholesterol supplementation resulted in a lower chance of mobile trajectories, whereas margaric acid incubation did not have a significant effect on PIEZO1 mobility. The “mobile” trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power-law model, revealing mobile PIEZO1 puncta exhibit anomalous subdiffusion. These studies illuminate the fundamental properties governing PIEZO1 diffusion in the plasma membrane and set the stage to determine how cellular processes and interactions may influence channel activity and mobility.

SIGNIFICANCE

PIEZO1 is a mechanically-activated ion channel that regulates a number of physiological processes. Here we examine a fundamental biophysical property of PIEZO1 - its movement in the plasma membrane. We find that the mobility of PIEZO1 is surprisingly heterogeneous, with some PIEZO1 puncta showing high mobility and some displaying very limited mobility. Cholesterol depletion from the plasma membrane increases PIEZO1 mobility while cholesterol supplementation decreases mobility. Yoda1 treatment increases PIEZO1 mobility whereas GsMTx-4 treatment decreases channel mobility.
An empirical study on the use of a facial emotion recognition system in guidance counseling utilizing the technology acceptance model and the general comfort questionnaire
Authors: Gom-os Dhong Fhel K. , Yong Kelvin Y.
Published: September 2022

Purpose

The goal of this study is to test the real-world use of an emotion recognition system.

Design/methodology/approach

The researchers chose an existing algorithm that displayed high accuracy and speed. Four emotions: happy, sadness, anger and surprise, are used from six of the universal emotions, associated by their own mood markers. The mood-matrix interface is then coded as a web application. Four guidance counselors and 10 students participated in the testing of the mood-matrix. Guidance counselors answered the technology acceptance model (TAM) to assess its usefulness, and the students answered the general comfort questionnaire (GCQ) to assess their comfort levels.

Findings

Results from TAM found that the mood-matrix has significant use for the guidance counselors and the GCQ finds that the students were comfortable during testing.

Originality/value

No study yet has tested an emotion recognition system applied to counseling or any mental health or psychological transactions.
Prioritization of risks associated with the implementation of project-based learning concept in engineering institutions
Authors: Pasi Bhaveshkumar Nandanram , Dongare Pallavi Vivek , Rawat Suman Joshi
Published: September 2022

Purpose

This research article aims to prioritize the risks associated with the implementation of the project-based learning (PBL) concept in engineering institutions and develop possible strategies for risk management.

Design/methodology/approach

In this research article, various risks associated with the implementation of the PBL concept in engineering institutions are discovered by taking inputs from academicians and performing a literature survey of peer-reviewed journal articles. Then, identified risks are prioritized by using the Technique for Order Preference by Similarity to the Ideal Solution (TOPSIS) method. Finally, the risk mitigation strategies are developed.

Findings

From the literature survey, 11 risks associated with the implementation of the PBL concept are identified. The TOPSIS method resulted in group dynamics risk and faculty training risk as the top two risks in the implementation of the PBL concept, whereas anxiety risk and poor prior learning experience risk are relatively low-ranked risks.

Research limitations/implications

The outcome of the research is based on the responses received through questionnaires. There are other methods also available for risk analysis, which are beyond this study.

Practical implications

The outcome of this research work will help the implementer of the PBL concept to effectively deal with the risks involved in implementing the PBL concept in engineering institutions by adopting strategies.

Originality/value

This research paper gives an idea about risks associated with the PBL implementation in engineering institutions. Also, this paper uses TOPSIS method for ranking of identified risks.
Remote sensing to assess the risk for cultural heritage: forecasting potential collapses due to rainfall in historic fortifications
Authors: Moreno Mónica , Ortiz Rocío , Ortiz Pilar
Published: September 2022

Purpose

Heavy rainfall is one of the main causes of the degradation of historic rammed Earth architecture. For this reason, ensuring the conservation thereof entails understanding the factors involved in these risk situations. The purpose of this study is to research three past events in which rainfall caused damage and collapse to historic rammed Earth fortifications in Andalusia in order to analyse whether it is possible to prevent similar situations from occurring in the future.

Design/methodology/approach

The three case studies analysed are located in the south of Spain and occurred between 2017 and 2021. The hazard presented by rainfall within this context has been obtained from Art-Risk 3.0 (Registration No. 201999906530090). The vulnerability of the structures has been assessed with the Art-Risk 1 model. To characterise the strength, duration, and intensity of precipitation events, a workflow for the statistical use of GPM and GSMaP satellite resources has been designed, validated, and tested. The strength of the winds has been evaluated from data from ground-based weather stations.

Findings

GSMaP precipitation data is very similar to data from ground-based weather stations. Regarding the three risk events analysed, although they occurred in areas with a torrential rainfall hazard, the damage was caused by non-intense rainfall that did not exceed 5 mm/hour. The continuation of the rainfall for several days and the poor state of conservation of the walls seem to be the factors that triggered the collapses that fundamentally affected the restoration mortars.

Originality/value

A workflow applied to vulnerability and hazard analysis is presented, which validates the large-scale use of satellite images for past and present monitoring of heritage structure risk situations due to rain.
Minority dissent, social acceptance and conflict transformation in multiparty systems
Authors: Curșeu Petru Lucian , Schruijer Sandra G.L.
Published: September 2022

Purpose

This study aims to investigate the role of minority dissent (MD) as an antecedent for task (TC) and relationship conflict (RC) in groups engaged in multiparty collaboration. The authors hypothesized that MD triggers both TC and RC and that the association between MD and RC is mediated by TC. Moreover, the authors hypothesized that the positive association between MD and RC is attenuated by social acceptance, while the positive association between TC and RC is attenuated by trust.

Design/methodology/approach

The authors have tested the hypotheses in 36 groups comprising in total 145 professionals that attended a two-day workshop on working across organizational boundaries and who filled in three surveys during a multiparty simulation. The authors used multilevel mediation analyses to test the hypotheses.

Findings

The results generally supported the role of MD as an antecedent for both TC and RC as well as the mediating role of TC in the relationship between MD and RC. The attenuating role of social acceptance in the relationship between MD and RC was fully supported, while the attenuating role of trust in the relationship between TC and RC was not supported.

Research limitations/implications

This study is based on a rather small sample and used a cross-lagged data collection design, and no causal claims can be derived from the findings. Behavioral multiparty simulations create a realistic context in which the authors investigate the dynamics of conflict transformation and explore the interplay of MD, TC and RC.

Social implications

As nowadays, multiparty systems are engaged in dealing with important societal challenges and because RC is detrimental for collaborative effectiveness, the results have important implications for facilitating effective collaboration in such complex systems.

Originality/value

This study makes an important contribution to the literature on conflict in multiparty systems by showing that as an antecedent of intragroup conflict, MD can have both a beneficial as well as a detrimental impact on the conflict dynamics of multiparty systems. It points out the importance of social acceptance as a buffer against the detrimental role of MD.