An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis.

Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation1,2. Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms3,4. One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes 5 . Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations.

Comparison of Whiskbroom and Pushbroom darkfield elastic light scattering spectroscopic imaging for head and neck cancer identification in a mouse model.

The early detection of head and neck cancer is a prolonged challenging task. It requires a precise and accurate identification of tissue alterations as well as a distinct discrimination of cancerous from healthy tissue areas. A novel approach for this purpose uses microspectroscopic techniques with special focus on hyperspectral imaging (HSI) methods. Our proof-of-principle study presents the implementation and application of darkfield elastic light scattering spectroscopy (DF ELSS) as a non-destructive, high-resolution, and fast imaging modality to distinguish lingual healthy from altered tissue regions in a mouse model. The main aspect of our study deals with the comparison of two varying HSI detection principles, which are a point-by-point and line scanning imaging, and whether one might be more appropriate in differentiating several tissue types. Statistical models are formed by deploying a principal component analysis (PCA) with the Bayesian discriminant analysis (DA) on the elastic light scattering (ELS) spectra. Overall accuracy, sensitivity, and precision values of 98% are achieved for both models whereas the overall specificity results in 99%. An additional classification of model-unknown ELS spectra is performed. The predictions are verified with histopathological evaluations of identical HE-stained tissue areas to prove the model's capability of tissue distinction. In the context of our proof-of-principle study, we assess the Pushbroom PCA-DA model to be more suitable for tissue type differentiations and thus tissue classification. In addition to the HE-examination in head and neck cancer diagnosis, the usage of HSI-based statistical models might be conceivable in a daily clinical routine.

Myosin 10 is involved in murine pigmentation.

Myosins are molecular motors that are well known for their role in cell movement and contractile functions. Although extensively studied in muscle physiology, little is known about the function of myosins in mammalian skin. As part of the Sanger Institute Mouse Genetics Project, we have identified a role for Myo10 in pigmentation, with a phenotype unlike those of Myo5a or Myo7a. Adult mice homozygous for a disrupted Myo10 allele on a C57BL/6N background displayed a high degree of penetrance for white patches on their abdomen and dorsal surface. Forepaw syndactyly and hind paw syndactyly were also observed in these mice. Tail epidermal wholemounts showed a complete lack of melanocytes in the hair follicles and interfollicular epidermis. Myo10 has previously been implicated in human pigmentation. Our current study reveals involvement of Myo10 in murine skin pigmentation.

Redefining the structure of the hair follicle by 3D clonal analysis.

The hair follicle (HF) is a multi-tissue mini-organ that self-renews periodically. However, the cellular organisation of this much-studied model is not fully understood. The structures of the outer layer and of the bulb, which ensures HF growth, have not been completely established. To clarify these points, we have conducted in vivo clonal analyses with 3D imaging in mice. The upper two-thirds of the HF outer layer consists of two clonally unrelated groups of cells that exhibit different modes of growth. They correspond to the basal outer root sheath (ORS) and the companion layer (Cp). The basal ORS has an unusual anisotropic mode of growth from a suprabulbar zone, which we named the privileged proliferation zone. The Cp has a stem/transient-amplifying mode of growth and is shown to be an HF internal structure. Furthermore, we describe an additional element, the bulb outer layer, which is contiguous and shares markers (e.g. Lgr5) with the basal ORS but is formed by a separate lineage that belongs neither to the ORS nor Cp lineage. It represents a novel element with proximal cells that are contiguous with the germinative layer in the bulb. In reference to its shape and position we named it the lower proximal cup (LPC). These clonal hierarchies reveal a novel model of HF organisation and growth based on two major entities: the basal ORS and the LPC plus the seven internal layers.

Cell-vision fusion: A Swin transformer-based approach for predicting kinase inhibitor mechanism of action from Cell Painting data.

Image-based profiling of the cellular response to drug compounds has proven effective at characterizing the morphological changes resulting from perturbation experiments. As data availability increases, however, there are growing demands for novel deep-learning methods. We applied the SwinV2 computer vision architecture to predict the mechanism of action of 10 kinase inhibitor compounds directly from Cell Painting images. This method outperforms the standard approach of using image-based profiles (IBP)-multidimensional feature set representations generated by bioimaging software. Furthermore, our fusion approach-cell-vision fusion, combining three different data modalities, images, IBPs, and chemical structures-achieved 69.79% accuracy and 70.56% F1 score, 4.20% and 5.49% higher, respectively, than the best-performing IBP method. We provide three techniques, specific to Cell Painting images, which enable deep-learning architectures to train effectively and demonstrate approaches to combat the significant batch effects present in large Cell Painting datasets.

Single-cell and spatially resolved interactomics of tooth-associated keratinocytes in periodontitis.

Periodontitis affects billions of people worldwide. To address relationships of periodontal niche cell types and microbes in periodontitis, we generated an integrated single-cell RNA sequencing (scRNAseq) atlas of human periodontium (34-sample, 105918-cell), including sulcular and junctional keratinocytes (SK/JKs). SK/JKs displayed altered differentiation states and were enriched for effector cytokines in periodontitis. Single-cell metagenomics revealed 37 bacterial species with cell-specific tropism. Fluorescence in situ hybridization detected intracellular 16 S and mRNA signals of multiple species and correlated with SK/JK proinflammatory phenotypes in situ. Cell-cell communication analysis predicted keratinocyte-specific innate and adaptive immune interactions. Highly multiplexed immunofluorescence (33-antibody) revealed peri-epithelial immune foci, with innate cells often spatially constrained around JKs. Spatial phenotyping revealed immunosuppressed JK-microniches and SK-localized tertiary lymphoid structures in periodontitis. Here, we demonstrate impacts on and predicted interactomics of SK and JK cells in health and periodontitis, which requires further investigation to support precision periodontal interventions in states of chronic inflammation.

Hair follicle renewal: authentic morphogenesis that depends on a complex progression of stem cell lineages.

The hair follicle (HF) grows during the anagen phase from precursors in the matrix that give rise to each differentiated HF layer. Little is known about the lineal relationship between these layer-restricted precursors and HF stem cells. To understand how the HF stem cells regenerate the typical anagen organization, we conducted in vivo clonal analysis of key stages of the HF cycle in mice. Unexpectedly, we found that the pool of HF stem cells contains precursors with both multipotent and restricted contributions. This implies that the lineal relationships between HF stem cells (persisting during telogen) and layer-restricted precursors (in the germinative layer), responsible for HF elongation during anagen, are not stereotyped. Formation of the matrix at each cycle is accompanied by the transient expansion of an intermediary pool of precursors at the origin of the germinative layer and by the progressive restriction of cell dispersion. The regionalization of clonal patterns within the outer HF structure (the outer root sheath) suggests that the position of the precursors might be a crucial factor in determining their fate. The presence of HF stem cells with multipotent contribution and the progressive segregation of HF lineages upon anagen activation indicate that each HF renewal cycle constitutes an authentic morphogenetic process. A comprehensive model was constructed based on the different clonal patterns observed. In this model, the positions of the precursors relative to the dermal papilla together with the progressive restriction of cell dispersion are part of the mechanism that restricts their contribution to the different HF lineages.

Myogenic waves and myogenic programs during Xenopus embryonic myogenesis.

UNLABELLED: Although Xenopus is a key model organism in developmental biology, little is known about the myotome formation in this species. Here, we assessed the expression of myogenic regulatory factors of the Myod family (MRFs) during embryonic development and revealed distinct MRF programs. RESULTS: The expression pattern of each MRF during embryonic development highlights three successive myogenic waves. We showed that a first median and lateral myogenesis initiates before dermomyotome formation: the median cell population expresses Myf5, Myod, and Mrf4, whereas the lateral one expresses Myod, moderate levels of Myogenin and Mrf4. The second wave of myoblasts arising from the dermomyotome is characterized by the full MRF program expression, with high levels of Myogenin. The third wave is revealed by Myf5 expression in the myotome and could contribute to the formation of plurinucleated fibers at larval stages. Furthermore, Myf5- or Myod-expressing anlagen are identified in craniofacial myogenesis. CONCLUSIONS: The first median and lateral myogenesis and their associated MRF programs have probably disappeared in mammals. However, some aspects of Xenopus myogenesis have been conserved such as the development of somitic muscles by successive myogenic waves and the existence of Myf5-dependent and -independent lineages.

Fat Graft Retention: Adipose Tissue, Adipose-Derived Stem Cells, and Aging.

SUMMARY: Over the past 30 years, there has been a dramatic increase in the use of autologous fat grafting for soft-tissue augmentation and to improve facial skin quality. Several studies have highlighted the impact of aging on adipose tissue, leading to a decrease of adipose tissue volume and preadipocyte proliferation and increase of fibrosis. Recently, there has been a rising interest in adipose tissue components, including adipose-derived stem/stromal cells (ASCs) because of their regenerative potential, including inflammation, fibrosis, and vascularization modulation. Because of their differentiation potential and paracrine function, ASCs have been largely used for fat grafting procedures, as they are described to be a key component in fat graft survival. However, many parameters as surgical procedures or adipose tissue biology could change clinical outcomes. Variation on fat grafting methods have led to numerous inconsistent clinical outcomes. Donor-to-donor variation could also be imputed to ASCs, tissue inflammatory state, or tissue origin. In this review, the authors aim to analyze (1) the parameters involved in graft survival, and (2) the effect of aging on adipose tissue components, especially ASCs, that could lead to a decrease of skin regeneration and fat graft retention. CLINICAL RELEVANCE STATEMENT: This review aims to enlighten surgeons about known parameters that could play a role in fat graft survival. ASCs and their potential mechanism of action in regenerative medicine are more specifically described.

Lrig1-expression confers suppressive function to CD4+ cells and is essential for averting autoimmunity via the Smad2/3/Foxp3 axis.

Regulatory T cells (Treg) are CD4+ T cells with immune-suppressive function, which is defined by Foxp3 expression. However, the molecular determinants defining the suppressive population of T cells have yet to be discovered. Here we report that the cell surface protein Lrig1 is enriched in suppressive T cells and controls their suppressive behaviors. Within CD4+ T cells, Treg cells express the highest levels of Lrig1, and the expression level is further increasing with activation. The Lrig1+ subpopulation from T helper (Th) 17 cells showed higher suppressive activity than the Lrig1- subpopulation. Lrig1-deficiency impairs the suppressive function of Treg cells, while Lrig1-deficient naïve T cells normally differentiate into other T cell subsets. Adoptive transfer of CD4+Lrig1+ T cells alleviates autoimmune symptoms in colitis and lupus nephritis mouse models. A monoclonal anti-Lrig1 antibody significantly improves the symptoms of experimental autoimmune encephalomyelitis. In conclusion, Lrig1 is an important regulator of suppressive T cell function and an exploitable target for treating autoimmune conditions.

Feline Oral Squamous Cell Carcinoma: A Critical Review of Etiologic Factors.

Feline oral squamous cell carcinoma (FOSCC) is the most common oral neoplasia in cats. This malignant tumor is locally invasive, has a high mortality rate, and its etiology is not yet known. In humans, head and neck squamous cell carcinoma is associated with tobacco smoke, alcohol consumption, and human papillomavirus infection. Herein, a critical review about the potential etiologic factors of FOSCC was performed, considering publications between 2000 and 2022, aiming to synthesize all available scientific evidence regarding this issue. Recommendations of the PRISMA statement and the Cochrane Collaboration were followed and the PubMed database searched by using the MeSH terms MeSH terms "oral", "mouth", "lingual", "labial", "gingiva", "carcinoma", "squamous", and "feline". The selection process for eligible studies was based on specific inclusion and exclusion criteria and the quality of the studies assessed. The initial search resulted in 553 publications, with only 26 of these being included in the review. Sixteen studies were related to viral etiology and nine related to environmental factors such as exposure to tobacco smoke, ectoparasitic products, and the presence of oral comorbidities. When evaluated, feline papillomavirus was detected in 16.2% of samples of FOSCC. In the three studies focused on exposure to tobacco smoke, 35.2% (30/85) of cats with FOSCC had a history of this exposure. The consumption of canned food and the use of deworming collars were associated, in only one publication, with a risk of neoplasia increased by 4.7 and 5.3 times, respectively. Among 485 cats with FOSCC, 6.4% had dental and oral pathology (i.e., periodontal disease or feline chronic gingivostomatitis). The present study demonstrates that the available evidence on the etiology of FOSCC is still limited, however, there has been an increasing interest on this topic. To better understand the role of the possible etiological factors of this aggressive disease, and model for its human counterpart, large, prospective multi-institutional studies are needed.

Assessing the Role of Carbonyl Adducts, Particularly Malondialdehyde Adducts, in the Development of Dermis Yellowing Occurring during Skin Photoaging.

Solar elastosis is associated with a diffuse yellow hue of the skin. Photoaging is related to lipid peroxidation leading to the formation of carbonyl groups. Protein carbonylation can occur by addition of reactive aldehydes, such as malondialdehyde (MDA), 4-hydroxy-nonenal (4-HNE), and acrolein. All the proteins concerned with this modification, and the biological consequences of adduct formation, are not completely identified. The link between yellowish skin and dermal carbonylated proteins induced by aldehyde adducts was investigated. The study was carried out on ex vivo skin samples from sun-exposed or sun-protected areas and on in vitro dermal equivalent models incubated with 5 mM MDA, 4-HNE, or acrolein. The yellow color and the level of MDA, 4-HNE, and acrolein adducts were evaluated. Yellowish color differences were detected in the dermis of sun-exposed skin compared to sun-protected skin and in in vitro models following addition of MDA, 4-HNE, or acrolein. The yellowing was correlated with the carbonyl adducts increasing in the dermis and in in vitro models incubated with aldehydes. The stronger yellowing seemed to be mediated more by MDA than 4-HNE and acrolein. These observations suggest that dermal carbonylation especially induced by MDA result in the yellow hue of dermis and is involved, in part, in the yellowing observed during skin photoaging.

A Scarless Healing Tale: Comparing Homeostasis and Wound Healing of Oral Mucosa With Skin and Oesophagus.

Epithelial tissues are the most rapidly dividing tissues in the body, holding a natural ability for renewal and regeneration. This ability is crucial for survival as epithelia are essential to provide the ultimate barrier against the external environment, protecting the underlying tissues. Tissue stem and progenitor cells are responsible for self-renewal and repair during homeostasis and following injury. Upon wounding, epithelial tissues undergo different phases of haemostasis, inflammation, proliferation and remodelling, often resulting in fibrosis and scarring. In this review, we explore the phenotypic differences between the skin, the oesophagus and the oral mucosa. We discuss the plasticity of these epithelial stem cells and contribution of different fibroblast subpopulations for tissue regeneration and wound healing. While these epithelial tissues share global mechanisms of stem cell behaviour for tissue renewal and regeneration, the oral mucosa is known for its outstanding healing potential with minimal scarring. We aim to provide an updated review of recent studies that combined cell therapy with bioengineering exporting the unique scarless properties of the oral mucosa to improve skin and oesophageal wound healing and to reduce fibrotic tissue formation. These advances open new avenues toward the ultimate goal of achieving scarless wound healing.

Preparation of biological samples for microplastic identification by Nile Red.

Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 µm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94-100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97-100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

The Xenopus MEF2 gene family: evidence of a role for XMEF2C in larval tendon development.

MEF2 transcription factors are well-established regulators of muscle development. In this report, we describe the cloning of multiple splicing isoforms of the XMEF2A and XMEF2C encoding genes, differentially expressed during Xenopus development. Using whole-mount in situ hybridization, we found that the accumulation of XMEF2C mRNA in the tadpole stages was restricted to intersomitic regions and to the peripheral edges of hypaxial and cranial muscle masses in contrast to XMEF2A and XMEF2D, characterized by a continuous muscle cell expression. The XMEF2C positive cells express the bHLH transcription factor, Xscleraxis, known as a specific marker for tendons. Gain of function experiments revealed that the use of a hormone-inducible XMEF2C construct is able to induce Xscleraxis expression. Furthermore, XMEF2C specifically cooperates with Xscleraxis to induce tenascin C and betaig-h3, two genes preferentially expressed in Xenopus larval tendons. These findings 1) highlight a previously unappreciated and specific role for XMEF2C in tendon development and 2) identify a novel gene transactivation pathway where MEF2C cooperates with the bHLH protein, Xscleraxis, to activate specific gene expression.

Worldwide contamination of fish with microplastics: A brief global overview.

Widespread contamination of microplastics may lead to internalization in fish. This literature review from March 2019 to March 2020 details that a median of 60% of fish, belonging to 198 species captured in 24 countries, contain microplastics in their organs. Carnivores species ingested more microplastics than omnivores. Only 14% of fish were from aquaculture. Most studies focused on digestive systems, with presence in other organs currently being insufficiently assessed. Based on this assessment, knowledge gaps that should be addressed in future studies were identified.

Genomic landscape and clonal architecture of mouse oral squamous cell carcinomas dictate tumour ecology.

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression.

The Molecular Anatomy of Mouse Skin during Hair Growth and Rest.

Skin homeostasis is orchestrated by dozens of cell types that together direct stem cell renewal, lineage commitment, and differentiation. Here, we use single-cell RNA sequencing and single-molecule RNA FISH to provide a systematic molecular atlas of full-thickness skin, determining gene expression profiles and spatial locations that define 56 cell types and states during hair growth and rest. These findings reveal how the outer root sheath (ORS) and inner hair follicle layers coordinate hair production. We found that the ORS is composed of two intermingling but transcriptionally distinct cell types with differing capacities for interactions with stromal cell types. Inner layer cells branch from transcriptionally uncommitted progenitors, and each lineage differentiation passes through an intermediate state. We also provide an online tool to explore this comprehensive skin cell atlas, including epithelial and stromal cells such as fibroblasts, vascular, and immune cells, to spur further discoveries in skin biology.

The role of keratins in modulating carcinogenesis via communication with cells of the immune system.

Keratins are intermediate filament proteins expressed by epithelial cells and provide mechanical support for diverse epithelia. In our recent study (Sequeira et al., Nat Comm 9(1):3437), we analysed the role of keratin 76 (Krt76) in inflammation and cancer. Krt76 is expressed throughout embryonic development in the differentiated epithelial layers of a subset of stratified epithelia including tongue, palate and stomach. It is significantly downregulated in human oral squamous cell carcinoma (OSCC), correlating strongly with poor prognosis. We have shown that Krt76-/- mice exhibit systemic inflammation with increased levels of circulating B cells, regulatory T cells and effector T cells. When mice are given a chemical carcinogen in the drinking water, tongue and gastric cancer formation is accelerated in Krt76-/- mutant mice. Our data suggest that the increased tumour susceptibility of Krt76-/- mice is in part due to the enhanced accumulation of regulatory T cells in the tumour microenvironment. Our results support the notion that keratins, in addition to their function as cytoskeletal components, regulate immunity and affect tumour susceptibility of epithelial cells.

Heterogeneity within Stratified Epithelial Stem Cell Populations Maintains the Oral Mucosa in Response to Physiological Stress.

Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.