Innovative super-resolution in spatial transcriptomics: a transformer model exploiting histology images and spatial gene expression.

Spatial transcriptomics technologies have shed light on the complexities of tissue structures by accurately mapping spatial microenvironments. Nonetheless, a myriad of methods, especially those utilized in platforms like Visium, often relinquish spatial details owing to intrinsic resolution limitations. In response, we introduce TransformerST, an innovative, unsupervised model anchored in the Transformer architecture, which operates independently of references, thereby ensuring cost-efficiency by circumventing the need for single-cell RNA sequencing. TransformerST not only elevates Visium data from a multicellular level to a single-cell granularity but also showcases adaptability across diverse spatial transcriptomics platforms. By employing a vision transformer-based encoder, it discerns latent image-gene expression co-representations and is further enhanced by spatial correlations, derived from an adaptive graph Transformer module. The sophisticated cross-scale graph network, utilized in super-resolution, significantly boosts the model's accuracy, unveiling complex structure-functional relationships within histology images. Empirical evaluations validate its adeptness in revealing tissue subtleties at the single-cell scale. Crucially, TransformerST adeptly navigates through image-gene co-representation, maximizing the synergistic utility of gene expression and histology images, thereby emerging as a pioneering tool in spatial transcriptomics. It not only enhances resolution to a single-cell level but also introduces a novel approach that optimally utilizes histology images alongside gene expression, providing a refined lens for investigating spatial transcriptomics.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Differential gene expression in layer 3 pyramidal neurons across 3 regions of the human cortical visual spatial working memory network.

Visual spatial working memory (vsWM) is mediated by a distributed cortical network composed of multiple nodes, including primary visual (V1), posterior parietal (PPC), and dorsolateral prefrontal (DLPFC) cortices. Feedforward and feedback information is transferred among these nodes via projections furnished by pyramidal neurons (PNs) located primarily in cortical layer 3. Morphological and electrophysiological differences among layer 3 PNs across these nodes have been reported; however, the transcriptional signatures underlying these differences have not been examined in the human brain. Here we interrogated the transcriptomes of layer 3 PNs from 39 neurotypical human subjects across 3 critical nodes of the vsWM network. Over 8,000 differentially expressed genes were detected, with more than 6,000 transcriptional differences present between layer 3 PNs in V1 and those in PPC and DLPFC. Additionally, over 600 other genes differed in expression along the rostral-to-caudal hierarchy formed by these 3 nodes. Moreover, pathway analysis revealed enrichment of genes in V1 related to circadian rhythms and in DLPFC of genes involved in synaptic plasticity. Overall, these results show robust regional differences in the transcriptome of layer 3 PNs, which likely contribute to regional specialization in their morphological and physiological features and thus in their functional contributions to vsWM.

Long noncoding RNA functionality in imprinted domain regulation.

Genomic imprinting is a parent-of-origin dependent phenomenon that restricts transcription to predominantly one parental allele. Since the discovery of the first long noncoding RNA (lncRNA), which notably was an imprinted lncRNA, a body of knowledge has demonstrated pivotal roles for imprinted lncRNAs in regulating parental-specific expression of neighboring imprinted genes. In this Review, we will discuss the multiple functionalities attributed to lncRNAs and how they regulate imprinted gene expression. We also raise unresolved questions about imprinted lncRNA function, which may lead to new avenues of investigation. This Review is dedicated to the memory of Denise Barlow, a giant in the field of genomic imprinting and functional lncRNAs. With her passion for understanding the inner workings of science, her indominable spirit and her consummate curiosity, Denise blazed a path of scientific investigation that made many seminal contributions to genomic imprinting and the wider field of epigenetic regulation, in addition to inspiring future generations of scientists.

Improving Access to Safe Anesthetic Care in Rural and Remote Communities in Affluent Countries.

Inadequate access to anesthesia and surgical services is often considered to be a problem of low- and middle-income countries. However, affluent nations, including Canada, Australia, and the United States, also face shortages of anesthesia and surgical care in rural and remote communities. Inadequate services often disproportionately affect indigenous populations. A lack of anesthesia care providers has been identified as a major contributing factor to the shortfall of surgical and obstetrical care in rural and remote areas of these countries. This report summarizes the challenges facing the provision of anesthesia services in rural and remote regions. The current landscape of anesthesia providers and their training is described. We also explore innovative strategies and emerging technologies that could better support physician-led anesthesia care teams working in rural and remote areas. Ultimately, we believe that it is the responsibility of specialist anesthesiologists and academic health sciences centers to facilitate access to high-quality care through partnership with other stakeholders. Professional medical organizations also play an important role in ensuring the quality of care and continuing professional development. Enhanced collaboration between academic anesthesiologists and other stakeholders is required to meet the challenge issued by the World Health Organization to ensure access to essential anesthesia and surgical services for all.

Nucleoporin 107, 62 and 153 mediate Kcnq1ot1 imprinted domain regulation in extraembryonic endoderm stem cells.

Genomic imprinting is a phenomenon that restricts transcription to predominantly one parental allele. How this transcriptional duality is regulated is poorly understood. Here we perform an RNA interference screen for epigenetic factors involved in paternal allelic silencing at the Kcnq1ot1 imprinted domain in mouse extraembryonic endoderm stem cells. Multiple factors are identified, including nucleoporin 107 (NUP107). To determine NUP107's role and specificity in Kcnq1ot1 imprinted domain regulation, we deplete Nup107, as well as Nup62, Nup98/96 and Nup153. Nup107, Nup62 and Nup153, but not Nup98/96 depletion, reduce Kcnq1ot1 noncoding RNA volume, displace the Kcnq1ot1 domain from the nuclear periphery, reactivate a subset of normally silent paternal alleles in the domain, alter histone modifications with concomitant changes in KMT2A, EZH2 and EHMT2 occupancy, as well as reduce cohesin interactions at the Kcnq1ot1 imprinting control region. Our results establish an important role for specific nucleoporins in mediating Kcnq1ot1 imprinted domain regulation.

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant.

Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

Atp2c2 Is Transcribed From a Unique Transcriptional Start Site in Mouse Pancreatic Acinar Cells.

Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc.

A role for chromatin topology in imprinted domain regulation.

Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.

Cocrystalline Copolyimides of Poly(ethylene 2,6-naphthalate).

Copolycondensation of N,N'-bis(2-hydroxyethyl)-biphenyl-3,4,3',4'-tetracarboxylic diimide (5-25 mol %) with bis(2-hydroxyethyl)-2,6-naphthalate affords a series of cocrystalline, poly(ethylene 2,6-naphthalate) (PEN)-based poly(ester imide)s. The glass transition temperature rises with the level of comonomer, from 118 °C for PEN itself to 148 °C for the 25% diimide copolymer. X-ray powder and fiber diffraction studies show that, when 5 mol % or more of diimide is present, the α-PEN crystal structure is replaced by a new crystalline phase arising from isomorphic substitution of biphenyldiimide for PEN residues in the polymer crystal lattice. This new phase is provisionally identified as monoclinic, C2/m, with two chains per unit cell, a = 10.56, b = 6.74, c = 13.25 Å, and ß = 143.0°.

Epigenetic regulation of genomic imprinting from germ line to preimplantation.

Genomic imprinting is an epigenetic process that distinguishes parental alleles, resulting in parent-specific expression of a gene or cluster of genes. Imprints are acquired during gametogenesis when genome-wide epigenetic remodeling occurs. These imprints must then be maintained during preimplantation development, when another wave of genome-wide epigenetic remodeling takes place. Thus, for imprints to persist as parent-specific epigenetic marks, coordinated factors and processes must be involved to both recognize an imprint and protect it from genome-wide remodeling. Parent-specific DNA methylation has long been recognized as a primary epigenetic mark demarcating a genomic imprint. Recent work has advanced our understanding of how and when parent-specific DNA methylation is erased and acquired in the germ line as well as maintained during preimplantation development. Epigenetic factors have also been identified that are recruited to imprinted regions to protect them from genome-wide DNA demethylation during preimplantation development. Intriguingly, asynchrony in epigenetic reprogramming appears to be a recurrent theme with asynchronous acquisition between male and female germ lines, between different imprinted genes, and between the two parental alleles of a gene. Here, we review recent advancements and discuss how they impact our current understanding of the epigenetic regulation of genomic imprinting.

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes.

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERß and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20-80 µm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.

Epigenetic mechanisms of genomic imprinting: common themes in the regulation of imprinted regions in mammals, plants, and insects.

Genomic imprinting is a form of epigenetic inheritance whereby the regulation of a gene or chromosomal region is dependent on the sex of the transmitting parent. During gametogenesis, imprinted regions of DNA are differentially marked in accordance to the sex of the parent, resulting in parent-specific expression. While mice are the primary research model used to study genomic imprinting, imprinted regions have been described in a broad variety of organisms, including other mammals, plants, and insects. Each of these organisms employs multiple, interrelated, epigenetic mechanisms to maintain parent-specific expression. While imprinted genes and imprint control regions are often species and locus-specific, the same suites of epigenetic mechanisms are often used to achieve imprinted expression. This review examines some examples of the epigenetic mechanisms responsible for genomic imprinting in mammals, plants, and insects.

The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster.

BACKGROUND: CTCF is a versatile zinc finger DNA-binding protein that functions as a highly conserved epigenetic transcriptional regulator. CTCF is known to act as a chromosomal insulator, bind promoter regions, and facilitate long-range chromatin interactions. In mammals, CTCF is active in the regulatory regions of some genes that exhibit genomic imprinting, acting as insulator on only one parental allele to facilitate parent-specific expression. In Drosophila, CTCF acts as a chromatin insulator and is thought to be actively involved in the global organization of the genome. RESULTS: To determine whether CTCF regulates imprinting in Drosophila, we generated CTCF mutant alleles and assayed gene expression from the imprinted Dp(1;f)LJ9 mini-X chromosome in the presence of reduced CTCF expression. We observed disruption of the maternal imprint when CTCF levels were reduced, but no effect was observed on the paternal imprint. The effect was restricted to maintenance of the imprint and was specific for the Dp(1;f)LJ9 mini-X chromosome. CONCLUSIONS: CTCF in Drosophila functions in maintaining parent-specific expression from an imprinted domain as it does in mammals. We propose that Drosophila CTCF maintains an insulator boundary on the maternal X chromosome, shielding genes from the imprint-induced silencing that occurs on the paternally inherited X chromosome. See commentary: http://www.biomedcentral.com/1741-7007/8/104.

The effect of acute hypoxia on excitability in the heart and the L-type calcium channel as a therapeutic target.

Acute hypoxia is induced during coronary occlusion or when oxygen supply does not meet demand and can trigger cardiac arrhythmia. Cardiac ion channels shape the action potential and excitability of the heart. Acute hypoxia regulates the function of cardiac ion channels including the L-type Ca(2+) channel that is the main route for Ca(2+)influx into cardiac myocytes and shapes the plateau phase of the action potential. This article will review the evidence for alteration of ion channel function during hypoxia as a result of modification of thiol groups by reactive oxygen species. The effect of acute hypoxia on cardiac excitability will be examined and how this can lead to life threatening arrhythmias with particular reference to the L-type Ca(2+) channel. Recent evidence indicates the L-type Ca(2+) channel is a suitable target for the development of drugs that can modify channel function during hypoxia or oxidative stress to prevent induction of arrhythmia or development of pathology.

Effect of temperature-induced reactive oxygen species production on excitation-contraction coupling in mammalian skeletal muscle.

1. Here we review evidence obtained recently by us indicating that the poor longevity of isolated mammalian skeletal muscle preparations at temperatures in the normal physiological range is related to the increased production of reactive oxygen species (ROS) in the resting muscle. 2. Temperature-induced ROS production increases markedly above 32 degrees C in isolated, resting skeletal muscle and is associated with the gradual and irreversible functional deterioration of the muscle. 3. The majority of the temperature-induced muscle ROS originates in the mitochondria and acts on various sites involved in excitation-contraction coupling.

O2(*-) production at 37 degrees C plays a critical role in depressing tetanic force of isolated rat and mouse skeletal muscle.

To find out whether the decrease in muscle performance of isolated mammalian skeletal muscle associated with the increase in temperature toward physiological levels is related to the increase in muscle superoxide (O(2)(*-)) production, O(2)(*-) released extracellularly by intact isolated rat and mouse extensor digitorum longus (EDL) muscles was measured at 22, 32, and 37 degrees C in Krebs-Ringer solution, and tetanic force was measured in both preparations at 22 and 37 degrees C under the same conditions. The rate of O(2)(*-) production increased marginally when the temperature was increased from 22 to 32 degrees C, but increased fivefold when the temperature was increased from 22 to 37 degrees C in both rat and mouse preparations. This increase was accompanied by a marked decrease in tetanic force after 30 min incubation at 37 degrees C in both rat and mouse EDL muscles. Tetanic force remained largely depressed after return to 22 degrees C for up to 120 min. The specific maximum Ca(2+)-activated force measured in mechanically skinned fibers after the temperature treatment was markedly depressed in mouse fibers but was not significantly depressed in rat muscle fibers. The resting membrane and intracellular action potentials were, however, significantly affected by the temperature treatment in the rat fibers. The effects of the temperature treatment on tetanic force, maximum Ca(2+)-activated force, and membrane potential were largely prevented by 1 mM Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a membrane-permeable superoxide dismutase mimetic, indicating that the increased O(2)(*-) production at physiological temperatures is largely responsible for the observed depression in tetanic force at 37 degrees C by affecting the contractile apparatus and plasma membrane.

Mitochondrial superoxide production in skeletal muscle fibers of the rat and decreased fiber excitability.

Mammalian skeletal muscles generate marked amounts of superoxide (O(2)(.-)) at 37 degrees C, but it is not well understood which is the main source of O(2)(.-) production in the muscle fibers and how this interferes with muscle function. To answer these questions, O(2)(.-) production and twitch force responses were measured at 37 degrees C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O(2)(.-) production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O(2)(.-) production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O(2)(.-) accounts for most of the O(2)(.-) measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37 degrees C. Muscle fiber excitability at 37 degrees C was greatly improved in the presence of a membrane permeant O(2)(.-) dismutase mimetic (Tempol), demonstrating a direct link between O(2)(.-) production in the mitochondria and muscle fiber performance. This implicates mitochondrial O(2)(.-) production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell.

The generation of cloned Drosophila melanogaster.

We report here the first successful use of embryonic nuclear transfer to create viable adult Drosophila melanogaster clones. Given the generation time, cost effectiveness, and relative ease of embryonic nuclear transplant in Drosophila, this method can provide an opportunity to further study the constraints on development imposed by transplanting determined or differentiated nuclei.