bluuebunny/crossref_metadata_2025 huggingface dataset:
Tendances en matière de migration et d’urbanisation en lien avec le bien-être des familles au Canada : Regard sur l’incapacité et les questions autochtones
Authors: Hilbrecht Margo , Keating Norah
Published: January 2024

Les discussions sur la migration et l’urbanisation au Canada et ailleurs dans le monde mettent généralement l’accent sur les réalités individuelles. On a ainsi tendance à négliger l’importance des disparités et des relations familiales. Or, l’omission de tenir compte des réseaux familiaux élargis a non seulement des conséquences sur le bien-être des personnes qui immigrent ou qui s’installent dans les milieux urbains, mais aussi sur celui des membres de la famille qu’ils laissent derrière eux. Au-delà de cet horizon individuel, les politiques liées à la migration se traduisent souvent par des initiatives qui visent l’ensemble de la population. On oublie en quelque sorte les familles vulnérables ou à risque – en raison de certains facteurs sanitaires et/ou démographiques – et leurs besoins particuliers. Il conviendrait donc de s’attarder davantage à leur expérience de la migration et de l’urbanisation afin que les décideurs et les fournisseurs de services soient en mesure de leur offrir des perspectives équitables et favorables à leur bien-être. Le présent article s’intéresse aux effets de la migration et de l’urbanisation au Canada sur le bien-être des familles, en portant une attention particulière à deux groupes de population plus vulnérables : les familles dont l’un des membres a une incapacité et les familles autochtones. Ces deux groupes connaissent davantage l’exclusion, c’est-à-dire qu’ils ont tendance à être systématiquement exclus, ignorés, voire exposés à des risques. Les familles autochtones sont marquées depuis longtemps par le colonialisme, le racisme et l’oppression (Saul, 2014), ce qui a eu de graves conséquences sur les familles et le sous-financement chronique des services de soutien, tels que les soins de santé, le logement et la protection de l’enfance (Gouvernement du Canada, 2018a; Commission de vérité et réconciliation du Canada, 2015). Les familles dont l’un des membres a une incapacité1 doivent être en mesure d’accéder facilement à des soins de santé et à des services connexes abordables qui répondent à leurs besoins, afin d’assurer le bien-être de l’ensemble des membres de la famille
Application Of Articulation Learning Model to The Eye Entrepreneurship Lessons in Class Xi Majoring In Industrial Electronics Engineering SMK Negeri 2 Sungai Penuh
Authors: Andri Yuliandi  , Ambiyar  , Mahesi Agni Zaus
Published: January 2024

Student learning outcomes are a benchmark for the success of the educational process, but not all learning outcomes are satisfactory, including in the Entrepreneurship subject because students are less active in the learning process. So research was carried out using the Articulation learning model in the Entrepreneurship subject in class This research uses experimental research methods. Which has an experimental class and a control class. Based on the results of this research, it is learning using the articulation learning model had an effect on the learning outcomes of class XI at SMK Negeri 2 Kota Sungai Full Department of industrial electronics engineering for the 2023/2024 academic year
Métodos Avanzados para Ventas y Operaciones: Gestión Predictiva con Excel, RStudio y Python
Authors: Andocilla Chicaiza Santiago Alejandro , Naranjo Vaca Myriam Johanna , Hidalgo Cajo Iván Mesias , Yasaca Pucuna Saul
Published: January 2024

Es un libro esencial para cualquier profesional que busque mejorar su capacidad de gestión en ventas y operaciones utilizando herramientas de análisis predictivo. Escrito por expertos en el campo, este libro ofrece una guía completa y accesible para aprovechar al máximo las capacidades de Excel, RStudio y Python en el ámbito de la gestión empresarial. Lo que hace que este libro sea especialmente valioso es su enfoque práctico y su capacidad para abordar tanto conceptos fundamentales como técnicas avanzadas. Los lectores encontrarán una introducción clara a las herramientas de análisis de datos más utilizadas en el mundo empresarial, así como ejemplos concretos de cómo aplicar estas técnicas para mejorar la toma de decisiones en ventas y operaciones. Los autores demuestran un profundo conocimiento de las aplicaciones prácticas de estas herramientas y presenta casos de estudio que muestran cómo pueden utilizarse en situaciones del mundo real. Además, el libro incluye ejercicios y ejemplos detallados que ayudan a los lectores a desarrollar sus habilidades de manera efectiva. Una característica destacada de este libro es su enfoque en la gestión predictiva, lo que significa que no solo se limita a analizar datos históricos, sino que también ofrece estrategias para prever tendencias futuras y tomar decisiones informadas en consecuencia. Esto es particularmente relevante en un mundo empresarial en constante evolución, donde la anticipación de cambios y la adaptación rápida son cruciales.
Pembinaan Soal Higher Order Thinking Skill (HOTS) IPA dengan KIT IPA pada Sekolah Dasar
Authors: Tri Aminingsih  , Saepulrohman Asep , Nabila Adinda Adriansyah Putri , Heliawati Leny
Published: January 2024

Pesatnya pendidikan Islam khususnya sekolah sunnah yang dibarengi biaya Pendidikan yang cukup tinggi membuat banyak siswa khususnya dengan ekonomi menengah ke bawah kesulitan mendapatkan Pendidikan yang berkualitas. Sekolah Dasar Bait Ta’limy Mutiara Umat diberikan KIT IPA berupa paket atau set alat dan bahan pembelajaran Ilmu Pengetahuan Alam yang bertujuan untuk meningkatkan minat dan pemahaman siswa terhadap Ilmu Pengetahuan Alam yang bisa berupa penyediaan bahan dan alat praktikum, alat peraga, buku atau modul pembelajaran, atau bahkan program pelatihan bagi guru-guru IPA yang ada di sekolah tersebut. KIT IPA ini bertujuan untuk membantu siswa dalam memahami prinsip-prinsip dasar ilmu pengetahuan alam, mengembangkan pemahaman siswa tentang fenomena alam, dan melatih keterampilan dalam berpikir ilmiah serta aplikasi ilmu pengetahuan dalam kehidupan sehari-hari. Pada saat pengabdian, dilakukan pembinaan kepada mitra khususnya guru yang mengampuh mata Pelajaran IPA untuk meningkatkan pembelajaran IPA dengan menggunakan alat-alat KIT IPA.
Моделирование теплопереноса при локальной конвекции на фоне вертикального течения в двухслойной системе «воздух – тепловыделяющая пористая среда»
Authors: Колчанова Екатерина Андреевна , Сагитов Рафиль Вафавич
Published: January 2024

Convective heat transfer in an air layer partially filled with a heat-generating granular porous medium is studied. There is a slow seepage of air through the layer in the vertical direction with a constant velocity. Equal temperatures are maintained at the outer solid permeable boundaries, and the heat source strength is constant within the porous sublayer and is proportional to the solid volume fraction. The permanent heat generation within the porous sublayer, combined with the vertical throughflow, causes a nonlinear thermal profile which is conducive for convection to occur. The Boussinesq approximation and Darcy's law are used to describe this convection. Numerical solution of the nonlinear convective problem is obtained on the basis of Newton's method. In the limiting case, the numerical data for the onset of convection are compared with the results of the earlier paper of the authors, where a linear stability theory and a method for constructing of the fundamental system of partial solution vectors have been applied, and with the data by other authors. The stationary regimes of local convection, which occurs in an “air – heat-generating porous medium-air” system over the basic vertical throughflow, and its effect on the heat transfer from the porous air sublayer with the growth of supercriticality are studied. It is shown that, depending on the velocity of the basic throughflow (the Peclet number), convection excitation can be both soft (due to supercritical pitchfork bifurcation) and hard (when the loss of stability of the basic throughflow is accompanied by subcritical pitchfork bifurcation that gives rise to an unstable secondary convective regime). This secondary regime is replaced by a stable tertiary convective regime with increasing supercriticality. It has been found that the total heat transfer rate for the upward basic throughflow exceeds that for the downward basic throughflow significantly, and that local convection at any direction of this throughflow increases the heat transfer rate in the system. An increase in the Nusselt number with the growth of supercriticality is recorded. However, a noticeable contribution of local convection to the total heat transfer is observed only when all values of the Pecklet number are negative and its positive values are lower than 2.
Об особенностях идентификации переменных термомеханических характеристик функционально-градиентного прямоугольника
Authors: Ватульян Александр Ованесович , Нестеров Сергей Анатольевич
Published: January 2024

The inverse thermoelastic problem of identification of the variable properties of a functionally graded rectangle is studied. Unsteady vibrations are excited by applying mechanical and thermal loads to the upper side of the rectangle. To solve the direct problem in Laplace transforms, the method of separation of variables and the shooting method for harmonics are used. Transformants are inverted by expanding the origin in terms of shifted Legendre polynomials. The method proposed for solving the direct problem is verified by comparison with a finite element solution. The influence of the laws of change of variable characteristics on the boundary physical fields is analyzed. The displacement components give additional information on the mechanical loading, and the temperature measured on the upper side of the rectangle over a certain time interval – on the thermal loading. Assuming that the additional information admits expansion in Fourier series, the two-dimensional inverse problem is reduced to one-dimensional problems for various harmonics. The solution of the obtained nonlinear inverse problems is carried out on the basis of an iterative process, at each stage of which, in order to find corrections for thermomechanical characteristics, systems of Fredholm integral equations of the 1st kind are solved. The possibility of simultaneous reconstruction of several characteristics is investigated. The results of computational experiments on the phased reconstruction of thermomechanical characteristics are presented. The influence of the thermomechanical coupling parameter on the results of the thermal stress coefficient reconstruction was clarified.
Novel majority carrier accumulation insulated gate bipolar transistor with Schottky junction
Authors: Duan Bao-Xing , Liu Yu-Lin , Tang Chun-Ping , Yang Yin-Tang ,   Key Laboratory of the Ministry of Education for Wide Band-Gap Semiconductor Materials and Devices, School of Microelectronics, Xidian University, Xi’an 710071, China
Published: January 2024

Insulated gate bipolar transistor (IGBT) is the core of modern power semiconductor device, and has been widely used due to its excellent electrical characteristics. A novel majority carrier accumulation mode IGBT with Schottky junction contact gate semiconductor layer (AC-SCG IGBT) is proposed and investigated by TCAD simulation in this article. When the AC-SCG IGBT is in the on-state, a forward bias is applied to the gate. Due to the very low forward voltage drop (VF) of the Schottky barrier diode, the potential of the gate semiconductor layer is almost equal to the gate potential, which can accumulate a large number of majority carrier electrons in the drift region. In addition to the electrons existing, these accumulated electrons increase the conductivity of the drift region, thus significantly reducing VF. Therefore, the doping concentration of the drift region is not limited by VF. The lightly doped drift region can make AC-SCG IGBT have a higher breakdown voltage (BV). Moreover, it also reduces the barrier capacitance in the turn-off process, thus the overall Miller capacitance is small, which can quickly turn off and reduce the turn-off time (Toff) and turn-off loss (Eoff). The simulation results indicate that at the BV of 600 V, the VF of 0.84 V for the proposed AC-SCG IGBT is reduced by 46.2% compared with that for the conventional IGBT (VF of 1.56 V). The Eoff of the AC-SCG IGBT (0.77 mJ/cm2) is reduced by 52.5% compared with that for the conventional IGBT (1.62 mJ/cm2), and the Toff (155.8–222.7 ns) is reduced by 30%. The contradiction between VF and Eoff is eliminated. In addition, the proposed AC-SCG IGBT has a better anti-latch-up capability and is coupled with its higher BV, so it has a larger forward biased safe operating area (FBSOA). The proposed novel structure meets the development requirements for future IGBT device performance, and has great significance for guiding the development of the power semiconductor device field.
Fuzzy coloring and total fuzzy coloring of various types of intuitionistic fuzzy graphs
Authors:   Sri Krishna Arts and Science College, Coimbatore, Buvaneswari R. , Revathy P. ,   Sri Krishna College of Engineering and Technology, Coimbatore
Published: January 2024

In this paper, fuzzy coloring and total fuzzy coloring of intuitionistic fuzzy graphs are introduced. The fuzzy chromatic number, fuzzy chromatic index, total fuzzy chromatic number and total fuzzy chromatic index of both vertices and edges in intuitionistic fuzzy graphs are defined and their properties are analysed with illustrations.
Hydrodynamics and multiscale order in confluent epithelia
Authors: Armengol-Collado Josep-Maria , Carenza Livio Nicola , Giomi Luca
Published: January 2024

We formulate a hydrodynamic theory of confluent epithelia: i.e. monolayers of epithelial cells adhering to each other without gaps. Taking advantage of recent progresses toward establishing a general hydrodynamic theory of p-atic liquid crystals, we demonstrate that collectively migrating epithelia feature both nematic (i.e. p = 2) and hexatic (i.e. p = 6) orders, with the former being dominant at large and the latter at small length scales. Such a remarkable multiscale liquid crystal order leaves a distinct signature in the system’s structure factor, which exhibits two different power-law scaling regimes, reflecting both the hexagonal geometry of small cells clusters and the uniaxial structure of the global cellular flow. We support these analytical predictions with two different cell-resolved models of epithelia – i.e. the self-propelled Voronoi model and the multiphase field model – and highlight how momentum dissipation and noise influence the range of fluctuations at small length scales, thereby affecting the degree of cooperativity between cells. Our construction provides a theoretical framework to conceptualize the recent observation of multiscale order in layers of Madin–Darby canine kidney cells and pave the way for further theoretical developments.
Calcium transients trigger switch-like discharge of prostaglandin E2 in an extracellular signal-regulated kinase-dependent manner
Authors: Watabe Tetsuya , Yamahira Shinya , Takakura Kanako , Thumkeo Dean , Narumiya Shuh , Matsuda Michiyuki , Terai Kenta
Published: January 2024

Prostaglandin E
2
(PGE
2
) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE
2
secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors we demonstrate that calcium transients in a single cell cause PGE
2
-mediated radial spread of PKA activation (RSPA) in neighboring cells. By
in vivo
imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE
2
discharge in response to the increasing cytoplasmic Ca
2+
concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and
in vivo
. Thus, the PGE
2
discharge is regulated temporally by calcium transients and ERK activity.
Vacuolar H+-ATPase Determines Daughter Cell Fates through Asymmetric Segregation of the Nucleosome Remodeling and Deacetylase Complex
Authors: Xie Zhongyun , Chai Yongping , Zhu Zhiwen , Shen Zijie , Zhao Zhiguang , Xiao Long , Du Zhuo , Ou Guangshuo , Li Wei
Published: January 2024

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in
Caenorhabditis elegans
. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H
+
–adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD’s asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.
Integrative analysis of DNA replication origins and ORC/MCM binding sites in human cells reveals a lack of overlap
Authors: Tian Mengxue , Wang Zhenjia , Su Zhangli , Shibata Etsuko , Shibata Yoshiyuki , Dutta Anindya , Zang Chongzhi
Published: January 2024

Based on experimentally determined average inter-origin distances of ∼100 kb, DNA replication initiates from ∼50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the Origin Recognition Complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and 5 ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ∼7.5 million union origins identified by all datasets, only 0.27% were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques (20,250 shared origins), suggesting extensive variability in origin usage and identification. 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF binding sites, G-quadruplex sites and activating histone marks, these overlaps are comparable or less than that of known Transcription Start Sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ∼13,000 reproducible ORC binding sites in human cancer cells, and only 4.5% were within 1 kb of the ∼11,000 union MCM2-7 binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast,
S. cerevisiae
. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.
Tracing the substrate translocation mechanism in P-glycoprotein
Authors: Gewering Theresa , Waghray Deepali , Parey Kristian , Jung Hendrik , Tran Nghi N.B. , Zapata Joel , Zhao Pengyi , Chen Hao , Januliene Dovile , Hummer Gerhard , Urbatsch Ina L. , Moeller Arne , Zhang Qinghai
Published: January 2024

P-glycoprotein (Pgp) is a prototypical ABC transporter of great biological and clinical significance that confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs
1–3
. Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds
4–9
, but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.
CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism
Authors: Xu Mengyuan , Neelands Torben , Powers Alexander S. , Liu Yan , Miller Steven D. , Pintilie Grigore , Bois J. Du , Dror Ron O. , Chiu Wah , Maduke Merritt
Published: January 2024

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl
-
-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl
-
-permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl
-
-permeation pathway.
Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition
Authors: Sarver Dylan C. , Garcia-Diaz Jean , Saqib Muzna , Riddle Ryan C. , Wong G. William
Published: January 2024

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes,
TMEM263
, encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the
Tmem263
gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first two weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted GH/IGF-1 axis, which is critical for longitudinal bone growth.
Tmem263
-null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in
Tmem263
-null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling a wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of
TMEM263
may potentially cause GH insensitivity and impair linear growth.
Auxin Exposure Disrupts Feeding Behavior and Fatty Acid Metabolism in Adult Drosophila
Authors: Fleck Sophie A. , Biswas Puja , DeWitt Emily D. , Knuteson Rebecca L. , Eisman Robert C. , Nemkov Travis , D’Alessandro Angelo , Tennessen Jason M. , Rideout Elizabeth J. , Weaver Lesley N.
Published: January 2024

The ease of genetic manipulation in
Drosophila melanogaster
using the
Gal4/UAS
system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on
Drosophila
development and survival in one wild-type strain. Here we show that auxin delays larval development in another widely-used fly strain, and that short- and long-term auxin exposure in adult
Drosophila
induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.
Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms
Authors: Bendzunas George N. , Byrne Dominic P , Shrestha Safal , Daly Leonard A , Oswald Sally O. , Katiyar Samiksha , Venkat Aarya , Yeung Wayland , Eyers Claire E , Eyers Patrick A , Kannan Natarajan
Published: January 2024

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity
in vitro
and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.
Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms
Authors: Bendzunas George N. , Byrne Dominic P , Shrestha Safal , Daly Leonard A , Oswald Sally O. , Katiyar Samiksha , Venkat Aarya , Yeung Wayland , Eyers Claire E , Eyers Patrick A , Kannan Natarajan
Published: January 2024

In eukaryotes, protein kinase signaling is regulated by a diverse array of post- translational modifications (PTMs). While regulation by activation segment phosphorylation in Ser/Thr kinases is well understood, relatively little is known about how oxidation of cysteine (Cys) amino acids modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity
in vitro
and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.
Tissue-resident NK cells support survival in pancreatic cancer through promotion of cDC1-CD8T activity
Authors: Go Simei , Demetriou Constantinos , Hughes Sophie , Lanfredini Simone , Valenzano Giampiero , Ferry Helen , Arbe-Barnes Edward , Sivakumar Shivan , Bashford-Rogers Rachael , Middleton Mark R. , Mukherjee Somnath , Morton Jennifer , Jones Keaton , O’Neill Eric
Published: January 2024

The immunosuppressive microenvironment in PDAC prevents tumor control but strategies to restore anti-cancer immunology, by increasing CD8 T cell activity, have not been successful. Here we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident NK (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypofunctional NKG2C
-ve
NK population that supports CD8 T cell involvement. We show an equivalent population in human PDAC cohorts that represents an adaptive-like immunomodulatory trNK-cell that similarly supports CD8 T cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5i/αPD1 and IR-induced damage as a novel therapeutic approach.
Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis
Authors: Ayra-Plasencia Jessel , Symington Lorraine , Machín Félix
Published: January 2024

The cohesin complex maintains sister chromatid cohesion from S phase to anaphase onset. Cohesin also plays roles in chromosome structure and DNA repair. At anaphase onset, the cohesin subunit Scc1 is cleaved to allow segregation in an orderly manner, although some residual cohesin subunits remain to maintain chromosome structure. Efficient DNA double-strand break (DSB) repair by homologous recombination (HR) with the sister chromatid also depends on cohesin. Here, we have tested whether residual cohesin is important during DSB repair in anaphase/telophase (late mitosis). Using the well-established MAT switching system, we first show that HR is molecularly functional in late mitosis, and then show that cohesin is required for its efficiency. During DSBs in late mitosis, the segregated sister loci get closer and have episodes of coalescence, which may favour repair by HR. Here, we also show that these cytological phenotypes also depend on cohesin. Finally, full-length Scc1 returns after DSBs in late mitosis, suggesting that
de novo
widespread cohesin complexes can support these retrograde behaviours.
Chemotherapy activates inflammasomes to cause inflammation-associated bone loss
Authors: Wang Chun , Kaur Khushpreet , Xu Canxin , Abu-Amer Yousef , Mbalaviele Gabriel
Published: January 2024

Chemotherapy is a widely used treatment for a variety of solid and hematological malignancies. Despite its success in improving the survival rate of cancer patients, chemotherapy causes significant toxicity to multiple organs, including the skeleton, but the underlying mechanisms have yet to be elucidated. Using tumor-free mouse models, which are commonly used to assess direct off-target effects of anti-neoplastic therapies, we found that doxorubicin caused massive bone loss in wild-type mice, a phenotype associated with increased number of osteoclasts, leukopenia, elevated serum levels of DAMPs (e.g., cell-free DNA and ATP) and cytokines (e.g., IL-1β and IL-18). Accordingly, doxorubicin activated the AIM2 and NLRP3 inflammasomes in macrophages and neutrophils, causing inflammatory cell death pyroptosis and NETosis, which correlated with its leukopenic effects. Moreover, the effects of this chemotherapeutic agent on cytokine secretion, cell demise, and bone loss were attenuated to various extent in conditions of AIM2 and/or NLRP3 insufficiency. Thus, we found that inflammasomes are key players in bone loss caused by doxorubicin, a finding that may inspire the development of a tailored adjuvant therapy that preserves the quality of this tissue in patients treated with this class of drugs.
Evolutionary adaptation of the chromodomain of the HP1-protein Rhino allows the integration of chromatin and DNA sequence signals
Authors: Baumgartner Lisa , Ipsaro Jonathan J. , Hohmann Ulrich , Handler Dominik , Schleiffer Alexander , Duchek Peter , Brennecke Julius
Published: January 2024

Members of the diverse heterochromatin protein 1 (HP1) family of proteins play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin binding patterns, presumably due to their interactions with various specificity factors. Here, we elucidate the molecular basis of the protein-protein interaction between the HP1 protein Rhino, a critical factor of the
Drosophila
piRNA pathway, and Kipferl, a DNA sequence-specific C
2
H
2
zinc finger protein and Rhino guidance factor. Through phylogenetic analyses, structure prediction, and
in vivo
genetics, we identify a single amino acid change within Rhino’s chromodomain, G31D, that does not affect H3K9me2/3 binding but abolishes the specific interaction between Rhino and Kipferl. Flies carrying the

rhino
G31D

mutation phenocopy
kipferl
mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of

rhino
G31D

flies. Thus, Rhino’s chromodomain serves as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.
Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation
Authors: Lazzarin Erika , Gradisch Ralph , Skopec Sophie MC , Alves da Silva Leticia , Sebastianelli-Schoditsch Chiara , Szöllősi Dániel , Maier Julian , Sucic Sonja , Roblek Marko , Kanner Baruch I , Sitte Harald H , Stockner Thomas
Published: January 2024

The human GABA transporter (GAT1) is a membrane transporter that mediates the reuptake of the neurotransmitter GABA from the synaptic cleft into neurons and glial cells. Dysregulation of the transport cycle has been associated with epilepsy and neuropsychiatric disorders, highlighting the crucial role of the transporter in maintaining homeostasis of brain GABA levels. GAT1 is a secondary active transporter that couples the movement of substrate to the simultaneous transport of sodium and chloride ions along their electrochemical gradients. Using MD simulations, we identified a novel sodium recruiting site at the entrance to the outer vestibule, which attracts positively charged ions and increases the local sodium concentration, thereby indirectly increasing sodium affinity. Mutations of negatively charged residues at the recruiting site slowed the binding kinetics, while experimental data revealed a change in sodium dependency of GABA uptake and a reduction of sodium affinity. Simulation showed that sodium displays a higher affinity for the sodium binding site NA2, which plays a role in stabilisation of the outward-open conformation. We directly show that the presence of a sodium ion bound to NA2 increases the stability of the closed inner gate and restrains motions of TM5. We find that sodium is only weakly bound to NA1 in the absence of GABA, while the presence of the substrate strengthens the interaction due to the completed ion coordinating shell, explaining cooperativity between GABA and sodium.
Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation
Authors: Lazzarin Erika , Gradisch Ralph , Skopec Sophie M.C. , da Silva Leticia Alves , Szöllősi Dániel , Maier Julian , Sucic Sonja , Kanner Baruch I. , Sitte Harald H. , Stockner Thomas
Published: January 2024

The human GABA transporter (GAT1) is a membrane transporter that mediates the reuptake of the neurotransmitter GABA from the synaptic cleft into neurons and glial cells. Dysregulation of the transport cycle has been associated with epilepsy and neuropsychiatric disorders, highlighting the crucial role of the transporter in maintaining homeostasis of brain GABA levels. GAT1 is a secondary active transporter that couples the movement of substrate to the simultaneous transport of sodium and chloride ions along their electrochemical gradients. Using MD simulations, we identified a novel sodium recruiting site at the entrance to the outer vestibule, which attracts positively charged ions and increases the local sodium concentration, thereby indirectly increasing sodium affinity. Mutations of negatively charged residues at the recruiting site slowed the binding kinetics, while experimental data revealed a change in sodium dependency of GABA uptake and a reduction of sodium affinity. Simulation showed that sodium displays a higher affinity for the sodium binding site NA2, which plays a role in the stabilisation of the outward-open conformation. We directly show that the presence of a sodium ion bound to NA2 increases the stability of the closed inner gate and restrains motions of TM5. We find that sodium is only weakly bound to NA1 in the absence of GABA, while the presence of the substrate strengthens the interaction due to the completed ion coordinating shell, explaining cooperativity of between GABA and sodium.
Pharmacologically inducing regenerative cardiac cells by small molecule drugs
Authors: Zhou Wei , He Kezhang , Wang Chiyin , Wang Pengqi , Wang Dan , Wang Bowen , Geng Han , Lian Hong , Ma Tianhua , Nie Yu , Ding Sheng
Published: January 2024

In contrast to lower organisms, adult mammals cannot regenerate damaged hearts through de-differentiation of cardiomyocytes (CMs) into cells with regenerative capacity. Development of an enabling condition to induce such naturally unavailable cells with potential to proliferate and differentiate into CMs, i.e., regenerative cardiac cells (RCCs), in mammals will provide new insights and tools for heart regeneration. Here, a two-compound combination (2C), CHIR99021 and A-485, was identified to robustly induce RCCs from human embryonic stem cell (hESC) derived TNNT2
+
CMs
in vitro
, which was confirmed by lineage tracing experiments. Functional analyses revealed that RCCs expressed a spectrum of genes essential for cardiogenesis and exhibited potential to become functional CMs, endothelial cells (ECs) and smooth muscle cells (SMCs). Consistent with the results in human cells, 2C-induced generation of RCCs were also observed in neonatal rats CMs
in vitro
and
in vivo
. Remarkably, administration of 2C can induce RCCs in adult mouse hearts and significantly improve survival and heart function in the mice undergoing myocardial infarction. Mechanistically, CHIR99021 is indispensable for transcriptional and epigenetic activation of genes essential for RCC, whereas A-485 mainly function to epigenetically down-regulate H3K27Ac and particularly H3K9Ac in CMs. Their combination specifically enhances both H3K27Ac and H3K9Ac on RCC genes, facilitating the establishment of RCC state dedifferentiated from CMs. Therefore, our findings demonstrated the feasibility and revealed the mechanisms of pharmacological induction of RCCs from endogenous CMs, which could offer a promising regenerative strategy to repair injured hearts.
Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2
Authors: Brotherton Deborah H. , Nijjar Sarbjit , Savva Christos G. , Dale Nicholas , Cameron Alexander D.
Published: January 2024

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO
2
. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO
2
. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.
A seven-sex species recognizes self and non-self mating-type via a novel protein complex
Authors: Yan Guanxiong , Ma Yang , Wang Yanfang , Zhang Jing , Cheng Haoming , Tan Fanjie , Wang Su , Zhang Delin , Xiong Jie , Yin Ping , Miao Wei
Published: January 2024

Although most species have two sexes, multisexual (or multi-mating type) species are also widespread. However, it is unclear how mating-type recognition is achieved at the molecular level in multisexual species. The unicellular ciliate Tetrahymena thermophila has seven mating types, which are determined by the MTA and MTB proteins. In this study, we found that both proteins are essential for cells to send or receive complete mating-type information, and transmission of the mating-type signal requires both proteins to be expressed in the same cell. We found that MTA and MTB form a mating-type recognition complex that localizes to the plasma membrane, but not to the cilia. Stimulation experiments showed that the mating-type-specific regions of MTA and MTB mediate both self- and non-self-recognition, indicating that T. thermophila uses a dual approach to achieve mating-type recognition. Our results suggest that MTA and MTB form an elaborate multifunctional protein complex that can identify cells of both self and non-self mating types in order to inhibit or activate mating, respectively.
SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle
Authors: Herneisen Alice L , Peters Michelle L , Smith Tyler A , Shortt Emily , Lourido Sebastian
Published: January 2024

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.
A nonlinear total variation based computed tomography (CT) image reconstruction method using gradient reinforcement
Authors: Ertas Metin
Published: January 2024

Compressed sensing-based reconstruction algorithms have been proven to be more successful than analytical or iterative methods for sparse computed tomography (CT) imaging by narrowing down the solution set thanks to its ability to seek a sparser solution. Total variation (TV), one of the most popular sparsifiers, exploits spatial continuity of features by restricting variation between two neighboring pixels in each direction as using partial derivatives. When the number of projections is much fewer than the one in conventional CT, which results in much less sampling rate than the minimum required one, TV may not provide satisfactory results. In this study, a new regularizer is proposed which seeks for a sparser solution by reinforcing the gradient of TV and empowering the spatial continuity of features. The experiments are done by using both analitical phantom and real human CT images and the results are compared with conventional, four-directional, and directional TV algorithms by using contrast-to-noise ratio (CNR), signal-to-noise ratio (SNR) and Structural Similarity Index (SSIM) metrics. Both quantitative and visual evaluations show that the proposed method is promising for sparse CT image reconstruction by reducing the background noise while preserving the features and edges.