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Eating disorders are typical diseases of adolescence and early adulthood. About 1-3% of female juveniles suffer from anorexia nervosa (AN) or bulimia nervosa (BN). Today AN is still the psychiatric disease with the highest mortality rate. The peri-operative mortality rate of patients suffering from AN is in the range up to 15%. The beginning of AN is a lingering process and the majority of patients show increasingly restrictive eating habits ending in cachexia. Patients are obsessed by the predominant idea of being obese in spite of having a significant underweight. Patients suffering from bulimia break the strict regimen by eating enormous amounts of high calorie food. Such eating attacks are followed by weight reducing measures, mostly vomiting. Most of the physical changes caused by AN are due to starvation and loss of weight. The most significant medical complications are alterations of the cardiovascular system accompanied by decreasing contractility of the heart, bradycardia, electrocardiographic changes as well as disequilibrium of electrolytic and water balance. Most of these symptoms can be reversed by putting on weight. [ABSTRACT FROM AUTHOR]

Patients with Charcot-Marie-Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination. [ABSTRACT FROM AUTHOR]

Circular RNA (circRNA) is covalently closed, single-stranded RNA produced by back-splicing. A few circRNAs have been implicated as functional; however, we lack understanding of pathways that are regulated by circRNAs. Here we generated a pooled short-hairpin RNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at different levels (ranging from low to high) in humans. We used this library for loss-of-function proliferation screens in a panel of 18 cancer cell lines from four tissue types harbouring mutations leading to constitutive activity of defined pathways. Both context-specific and non-specific circRNAs were identified. Some circRNAs were found to directly regulate their precursor, whereas some have a function unrelated to their precursor. We validated these observations with a secondary screen and uncovered a role for circRERE(4-10) and circHUWE1(22,23), two cell-essential circRNAs, circSMAD2(2-6), a WNT pathway regulator, and circMTO1(2,RI,3), a regulator of MAPK signalling. Our work sheds light on pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Alpha, beta, and gamma tubulins are essential building blocks for all eukaryotic cells. The functions of the non-canonical tubulins, delta, epsilon, and zeta, however, remain poorly understood and their requirement in mammalian development untested. Herein we have used a spermatogenesis model to define epsilon tubulin (TUBE1) function in mice. We show that TUBE1 is essential for the function of multiple complex microtubule arrays, including the meiotic spindle, axoneme and manchette and in its absence, there is a dramatic loss of germ cells and male sterility. Moreover, we provide evidence for the interplay between TUBE1 and katanin-mediated microtubule severing, and for the sub-specialization of individual katanin paralogs in the regulation of specific microtubule arrays., (© 2024. The Author(s).)

The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate., (© 2023. The Author(s).)

The outbreak of SARS-CoV-2 has emphasized the need for a deeper understanding of infectivity, spread, and treatment of airborne viruses. Bacteriophages (phages) serve as ideal surrogates for respiratory pathogenic viruses thanks to their high tractability and the structural similarities tailless phages bear to viral pathogens. However, the aerosolization of enveloped SARS-CoV-2 surrogate phi6 usually results in a >3-log 10 reduction in viability, limiting its usefulness as a surrogate for aerosolized coronavirus in "real world" contexts, such as a sneeze or cough. Recent work has shown that saliva or artificial saliva greatly improves the stability of viruses in aerosols and microdroplets relative to standard dilution/storage buffers like suspension medium (SM) buffer. These findings led us to investigate whether we could formulate media that preserves the viability of phi6 and other phages in artificially derived aerosols. Results indicate that SM buffer supplemented with bovine serum albumin (BSA) significantly improves the recovery of airborne phi6, MS2, and 80α and outperforms commercially formulated artificial saliva. Particle sizing and acoustic particle trapping data indicate that BSA supplementation dose-dependently improves viral survivability by reducing the extent of particle evaporation. These data suggest that our viral preservation medium may facilitate a lower-cost alternative to artificial saliva for future applied aerobiology studies. IMPORTANCE We have identified common and inexpensive lab reagents that confer increased aerosol survivability on phi6 and other phages. Our results suggest that soluble protein is a key protective component in nebulizing medium. Protein supplementation likely reduces exposure of the phage to the air-water interface by reducing the extent of particle evaporation. These findings will be useful for applications in which researchers wish to improve the survivability of these (and likely other) aerosolized viruses to better approximate highly transmissible airborne viruses like SARS-CoV-2.

Mammalian preimplantation embryogenesis depends on the spatio-temporal dynamics of the microtubule cytoskeleton to enable exceptionally fast changes in cell number, function, architecture, and fate. Microtubule organising centres (MTOCs), which coordinate the remodelling of microtubules, are therefore of fundamental significance during the first days of a new life. Despite its indispensable role during early mammalian embryogenesis, the origin of microtubule growth remains poorly understood. In this review, we summarise the most recent discoveries on microtubule organisation and function during early human embryogenesis and compare these to innovative studies conducted in alternative mammalian models. We emphasise the differences and analogies of centriole inheritance and their role during the first cleavage. Furthermore, we highlight the significance of non-centrosomal MTOCs for embryo viability and discuss the potential of novel in vitro models and light-inducible approaches towards unravelling microtubule formation in research and assisted reproductive technologies., Competing Interests: Conflicts of interest None declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of β-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

With the advancement of cutting-edge live imaging technologies, microtubule remodelling has evolved as an integral regulator for the establishment of distinct differentiated cells. However, despite their fundamental role in cell structure and function, microtubules have received less attention when unravelling the regulatory circuitry of pluripotency. Here, we summarise the role of microtubule organisation and microtubule-dependent events required for the formation of pluripotent cells in vivo by deciphering the process of early embryogenesis: from fertilisation to blastocyst. Furthermore, we highlight current advances in elucidating the significance of specific microtubule arrays in in vitro culture systems of pluripotent stem cells and how the microtubule cytoskeleton serves as a highway for the precise intracellular movement of organelles. This Review provides an informed understanding of the intrinsic role of subcellular architecture of pluripotent cells and accentuates their regenerative potential in combination with innovative light-inducible microtubule techniques., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Delta (δ-) and epsilon (ε-) tubulin are lesser-known cousins of alpha (α-) and beta (β-) tubulin. They are likely to regulate centriole function in a broad range of species; however, their in vivo role and mechanism of action in mammals remain mysterious. In unicellular species and mammalian cell lines, mutations in δ- and ε-tubulin cause centriole destabilization and atypical mitosis and, in the most severe cases, cell death. Beyond the centriole, δ- and ε-tubulin localize to the manchette during murine spermatogenesis and interact with katanin-like 2 (KATNAL2), a protein with microtubule (MT)-severing properties, indicative of novel non-centriolar functions. Herein we summarize the current knowledge surrounding δ- and ε-tubulin, identify pathways for future research, and highlight how and why spermatogenesis and embryogenesis are ideal systems to define δ- and ε-tubulin function in vivo., Competing Interests: Declaration of interests The authors declare no interests., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development 1-4 . However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.

The preimplantation mouse embryo is a simple self-contained system, making it an excellent model to discover how mammalian cells function in real time and in vivo. Work over the last decade has revealed some key morphogenetic mechanisms that drive early development, yielding rudimentary instructions for the generation of a mammalian embryo. Here, we review the instructions revealed thus far, and then discuss remaining challenges to discover upstream factors controlling cell fate determination, test the role of mechanisms based on biological noise, and take advantage of recent technological developments to advance the spatial and temporal resolution of our current understanding., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The protein component of the myelin layer is essential for all aspects of peripheral nerves, and its deficiency can lead to structural and functional impairment. The presence of peripheral myelin protein 2 (P2, PMP2, FABP8, M-FABP) in Schwann cells has been known for decades and shown recently to be involved in the lipid homeostasis in the peripheral neural system. However, its precise role during de- and remyelination has yet to be elucidated. To this end, we assessed remyelination after sciatic nerve crush injury in vivo, and in an experimental de/remyelination ex vivo myelinating culture model in P2-deficient (P2 -/- ) and wild-type (WT) animals. In vivo, the nerve crush paradigm revealed temporal structural and functional changes in P2 -/- mice as compared to WT animals. Concomitantly, P2 -/- DRG cultures demonstrated the presence of shorter internodes and enlarged nodes after ex vivo de/remyelination. Together, these data indicate that P2 may play a role in remyelination of the injured peripheral nervous system, presumably by affecting the nodal and internodal configuration.

Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.

During preimplantation development, cells of the mammalian embryo must resolve their shape and position to ensure the future viability of the fetus. These initial changes are established as the embryo expands from one to thirty-two cells, and a group of originally spherical cells is transformed into a more polarized structure with distinct cell geometries and lineages. Recent advances in the application of non-invasive imaging technologies have enabled the discovery of mechanisms regulating patterning of the early mammalian embryo. Here, we review recent findings revealing cell protrusions that trigger early changes in cell shape and embryo compaction, and how anisotropies in mechanical forces drive the first spatial segregation of cells in the embryo to form the pluripotent inner mass., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

The microtubule cytoskeleton is critical for the generation and maturation of neurons in the developing mammalian nervous system. We have previously shown that mutations in the β-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities in humans. While it is known that TUBB5 is necessary for the proper generation and migration of neurons, little is understood of the role it plays in neuronal differentiation and connectivity. Here, we report that perturbations to TUBB5 disrupt the morphology of cortical neurons, their neuronal complexity, axonal outgrowth, as well as the density and shape of dendritic spines in the postnatal murine cortex. The features we describe are consistent with defects in synaptic signaling. Cellular-based assays have revealed that TUBB5 substitutions have the capacity to alter the dynamic properties and polymerization rates of the microtubule cytoskeleton. Together, our studies show that TUBB5 is essential for neuronal differentiation and dendritic spine formation in vivo, providing insight into the underlying cellular pathology associated with TUBB5 disease states., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)