OBJECTIVE: Atopic dermatitis (AD) is characterized by compositional and structural changes to the skin at lesional sites. Alteration to the levels and organization of both protein and lipid components are associated with disease status and lead to impaired barrier and hydration. Corneodesmosin (CDSN) and the arrangement and length of the intercellular lipid lamellae (ICLL) are altered in disrupted skin states. The aim of this research was to profile the distribution of CDSN and the ICLL in the stratum corneum (SC) at lesional and non-lesional sites in AD-prone skin and to investigate the impact of an eczema calming lotion containing petroleum jelly, fatty acids, and colloidal oatmeal.
METHODS: An IRB-approved study was conducted with participants with active AD. From a small subset of participants, tape strips were collected from lesional and non-lesional sites on the arm, prior to and after twice daily application, over 4 weeks of an eczema calming lotion containing petroleum jelly, fatty acids, and colloidal oatmeal. Fluorescent antibody staining was used to investigate the distribution of CDSN. Transmission electron microscopy (TEM) was used to characterize the ICLL.
RESULTS: The distribution/coverage of CDSN was similar between lesional and non-lesional sites at baseline; application of the lotion resulted in a more defined honeycomb/peripheral distribution. Normalized ICLL (nICLL) was lower in baseline samples from lesional sites relative to non-lesional sites. Application of the lotion increased this parameter by the end of the study at all sites.
CONCLUSIONS: The eczema calming lotion containing petroleum jelly, fatty acids and colloidal oatmeal provided changes in corneodesmosomal proteins distribution and ICLL, consistent with improvements in corneocyte maturation and improved barrier function in the skin of individuals with atopic dermatitis.
OBJECTIVE: La dermatite atopique (DA) est caractérisée par des modifications de la composition et de la structure de la peau au niveau des sites lésionnels. L\'altération des taux et de l\'organisation des composants protéiques et lipidiques est associée au statut de la maladie, et entraîne une altération de la barrière et de l\'hydratation. La cornéodesmosine (CDSN), et la disposition et la longueur des lamelles lipidiques intercellulaires (LLIC) sont altérées dans les états cutanés perturbés. L\'objectif de cette étude était d\'établir le profil de la distribution de la CDSN et des LLIC dans la couche cornée (CC) au niveau des sites lésionnels et non lésionnels dans la peau sujette à la DA, et d\'étudier l\'impact d\'une lotion apaisante contre l\'eczéma contenant de la vaseline, des acides gras et de l\'avoine colloïdale. MÉTHODES: Une étude approuvée par un CPP a été menée auprès de participants atteints de DA active. Dans un petit sous‐ensemble de participants, des bandes adhésives ont été prélevées sur des sites lésionnels et non lésionnels du bras, avant et après l\'application deux fois par jour pendant 4 semaines d\'une lotion apaisante contre l\'eczéma contenant de la vaseline, des acides gras et de l\'avoine colloïdale. Une coloration par anticorps fluorescents a été utilisée pour étudier la distribution de la CDSN. La microscopie électronique en transmission (MET) a été utilisée pour caractériser les LLIC. RÉSULTATS: La distribution/couverture de la CDSN était similaire entre les sites lésionnels et non lésionnels à l\'entrée dans l\'étude; l\'application de la lotion a entraîné une distribution en nid d\'abeille/périphérique plus définie. Le taux normalisé de LLIC (LLICn) était plus faible dans les échantillons prélevés à l\'entrée dans l\'étude au niveau des sites lésionnels par rapport aux sites non lésionnels. L\'application de la lotion a augmenté ce paramètre à la fin de l\'étude pour tous les sites.
CONCLUSIONS: La lotion apaisante contre l\'eczéma contenant de la vaseline, des acides gras et de l\'avoine colloïdale a entraîné des changements dans la distribution des protéines cornéodesmosomales et des LLIC, ce qui correspond à des améliorations de la maturation des cornéocytes et de la fonction de barrière de la peau des personnes atteintes de dermatite atopique.

Immunofluorescence microscopy is a powerful technique using fluorescently labelled antibodies which can be used to visualize proteins in the nucleus. A key advantage of this method is that it can provide insight into the spatial organization and the localization of nuclear proteins, which can provide elucidation of their function. Here, we provide a protocol for immunofluorescence staining in the nucleus, which has successfully been used to visualize histone modifications and nuclear bodies in human and mouse B lymphocytes, using as few as 1 × 104-5 × 104 cells.

UNASSIGNED: Prior studies suggested that neuromuscular factors might be involved in the pathogenesis of adolescent idiopathic scoliosis (AIS). The neuromuscular junction (NMJ) is the important pivot where the nervous system interacts with muscle fibers, but it has not been well characterized in the paraspinal muscles of AIS. This study aims to perform the quantitative morphological analysis of NMJs from paraspinal muscles of AIS.
UNASSIGNED: AIS patients who received surgery in our center were prospectively enrolled. Meanwhile, age-matched congenital scoliosis (CS) and non-scoliosis patients were also included as controls. Fresh samples of paraspinal muscles were harvested intraoperatively. NMJs were immunolabeled using different antibodies to reveal pre-synaptic neuronal architecture and post-synaptic motor endplates. A confocal microscope was used to acquire z-stack projections of NMJs images. Then, NMJs images were analyzed on maximum intensity projections using ImageJ software. The morphology of NMJs was quantitatively measured by a standardized \'NMJ-morph\' workflow. A total of 21 variables were measured and compared between different groups.
UNASSIGNED: A total of 15 AIS patients, 10 CS patients and 5 normal controls were enrolled initially. For AIS group, NMJs in the convex side of paraspinal muscles demonstrated obviously decreased overlap when compared with the concave side (34.27% ± 8.09% vs. 48.11% ± 10.31%, p = 0.0036). However, no variables showed statistical difference between both sides of paraspinal muscles in CS patients. In contrast with non-scoliosis controls, both sides of paraspinal muscles in AIS patients demonstrated significantly smaller muscle bundle diameters.
UNASSIGNED: This study first elucidated the morphological features of NMJs from paraspinal muscles of AIS patients. The NMJs in the convex side showed smaller overlap for AIS patients, but no difference was found in CS. This proved further evidence that neuromuscular factors might contribute to the mechanisms of AIS and could be considered as a novel potential therapeutic target for the treatment of progressive AIS.

This study investigates NADPH oxidase 4 (NOX4) involvement in iron-mediated astrocyte cell death in Alzheimer\'s Disease (AD) using single-cell sequencing data and transcriptomes. We analyzed AD single-cell RNA sequencing data, identified astrocyte marker genes, and explored biological processes in astrocytes. We integrated AD-related chip data with ferroptosis-related genes, highlighting NOX4. We validated NOX4\'s role in ferroptosis and AD in vitro and in vivo. Astrocyte marker genes were enriched in AD, emphasizing their role. NOX4 emerged as a crucial player in astrocytic ferroptosis in AD. Silencing NOX4 mitigated ferroptosis, improved cognition, reduced Aβ and p-Tau levels, and alleviated mitochondrial abnormalities. NOX4 promotes astrocytic ferroptosis, underscoring its significance in AD progression.

CONCLUSIONS: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

UNASSIGNED: Dental pulp stem cells (DPSCs) exhibit versatile differentiation capabilities, including neural differentiation, prompting the hypothesis that they may be implicated in the neurodevelopment of teeth. This study aimed to explore the temporospatial dynamics between DPSCs and tooth innervation, employing immunofluorescence staining and fluorescent dye injections to investigate the distribution of DPSCs, neural stem cells (NSCs), nerve growth cones, and sensory nerves in developing mouse tooth germs at various stages.
UNASSIGNED: Immunofluorescence staining targeting CD146, Nestin, and GAP-43, along with the injection of AM1-43 fluorescent dye, were utilized to observe the distribution of DPSCs, NSCs, nerve growth cones, and sensory nerves in mouse tooth germs at different developmental stages.
UNASSIGNED: Positive CD146 immunostaining was observed in microvascular endothelial cells and pericytes within and around the tooth germ. The percentage of CD146-positive cells remained consistent between 4-day-old and 8-day-old second molar tooth germs. Conversely, Nestin expression in odontoblasts and their processes decreased in 8-day-old tooth germs compared to 4-day-old ones. Positive immunostaining for GAP-43 and AM1-43 fluorescence revealed the entry of nerve growth cones and sensory nerves into the pulp in 8-day-old tooth germs, while these elements were confined to the dental follicle in 4-day-old germs. No co-localization of CD146-positive DPSCs with nerve growth cones and sensory nerves was observed.
UNASSIGNED: DPSCs and NSCs were present in dental pulp tissue before nerves penetrated the pulp. The decline in NSCs after nerve entry suggests a potential role for DPSCs and NSCs in attracting neural growth and/or differentiation within the pulp.

Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), β-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes.

This was an ambispective cohort study evaluating the prognostic significance of lymphocytic foci and its lymphoid composition in minor salivary gland biopsy (MSGB) for short-term disease flare and severity in Sjögren\'s syndrome (SS).
The inclusion criteria comprised individuals meeting the ACR/EULAR 2016 criteria who underwent MSGB with an infiltration of more than 50 lymphocytes and received clinical diagnosis between September 2017 and December 2018. Patients with inadequate biopsy samples were excluded. The number of lymphocytic foci and their lymphoid composition in MSGB were assessed using immunofluorescence staining. Major organ damage and improvements in the EULAR Sjögren\'s Syndrome Disease Activity Index (ESSDAI) were measured. Statistical analyses, including Cox and linear regressions, were conducted.
A total of 78 patients with at least one lymphocytic focus were included in the study. The presence of higher T-cell counts in lymphocytic foci in MSGB was associated with severe disease flare, and a logarithmic transformation of T-cell count indicated increased risk (HR 1.96, 95% CI 0.91-4.21). Improvements in the ESSDAI were associated with higher total lymphocyte count and T- and B-cell numbers in the lymphoid composition of the lymphocytic foci. Seropositive patients exhibited higher T CD4+ cell numbers. Correlation analysis showed negative associations between age and lymphocytic foci and the T-cell count. Positive correlations were observed between antinuclear antibody (ANA) titers and total lymphocyte numbers.
Patients with a higher number of T cells in the lymphocytic infiltrates of lymphocytic foci may have a two-fold risk of severe disease flare. The number of B cells and T CD4+ cells in the lymphocytic infiltrates of lymphocytic foci showed a weak but positive relation with the ESSDAI improvement during follow-up. Age and seropositivity appeared to influence the lymphoid composition of the lymphocytic foci.

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.