An adipocentric perspective on the development and progression of non-alcoholic fatty liver disease.

Alongside the liver, white adipose tissue (WAT) is critical in regulating systemic energy homeostasis. Although each organ has its specialised functions, they must work coordinately to regulate whole-body metabolism. Adipose tissues and the liver are relatively resilient and can adapt to an energy surplus by facilitating triglyceride (TG) storage up to a certain threshold level without significant metabolic disturbances. However, lipid storage in WAT beyond a "personalised" adiposity threshold becomes dysfunctional, leading to metabolic inflexibility, progressive inflammation, and aberrant adipokine secretion. Moreover, the failure of adipose tissue to store and mobilise lipids results in systemic knock-on lipid overload, particularly in the liver. Factors contributing to hepatic lipid overload include lipids released from WAT, dietary fat intake, and enhanced de novo lipogenesis. In contrast, extrahepatic mechanisms counteracting toxic hepatic lipid overload entail coordinated compensation through oxidation of surplus fatty acids in brown adipose tissue and storage of fatty acids as TGs in WAT. Failure of these integrated homeostatic mechanisms leads to quantitative increases and qualitative alterations to the lipidome of the liver. Initially, hepatocytes preferentially accumulate TG species leading to a relatively "benign" non-alcoholic fatty liver. However, with time, inflammatory responses ensue, progressing into more severe conditions such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, in some individuals (often without an early prognostic clue). Herein, we highlight the pathogenic importance of obesity-induced "adipose tissue failure", resulting in decreased adipose tissue functionality (i.e. fat storage capacity and metabolic flexibility), in the development and progression of NAFL/NASH.

Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium.

We investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early-stage egg chambers. We found that cell division is not required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. We examined the orientation of cell divisions with respect to the planar tissue axis and found a bias toward the primary direction of tissue expansion. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes-apical-basal and planar-is controlled by distinct, independent mechanisms in a proliferating epithelium.

Ahead of her time: The multidimensional impact of Marie Ussing Nylen.

Marie Ussing Nylen was a trail blazing scientist and administrative leader at the US National Institutes of Health. She accomplished this when it was extremely difficult for a woman to do so. She was also a whole person - a wife, mother, and talented athlete, that is, a well-rounded person by any definition. She was a gift to dental and oral science, as well as to those fortunate enough to know and work with her.

Immediate Implant and Customized Healing Abutment Promotes Tissues Regeneration: A 5-Year Clinical Report.

Significant alveolar bone resorption follows tooth extraction. Immediate implant placement alone is not sufficient to prevent this phenomenon. The present study aims at reporting the clinical and radiologic outcome of an immediate implant with a custom healing abutment. In this clinical case, a fractured upper first premolar was replaced by an immediate implant and a customized healing abutment designed on the perimeter of the extractive alveolus. After 3 months, the implant was restored. The facial and interdental soft tissue was maintained with appreciable success after 5 years. The pre-and 5-year post-treatment computerized tomography scans showed bone regeneration of the buccal plate. Use of an interim customized healing abutment helps prevent hard- and soft-tissue collapse and promotes bone regeneration. This technique is straightforward and may represent a smart preservation strategy when there is no indication for adjunctive hard or soft tissue grafting. Given the limited nature of this case report, further studies are needed to confirm the present findings.

Immediate Implant and Customized Healing Abutment Promotes Tissues Regeneration: a 5-year Clinical Report.

INTRODUCTION: Significant alveolar bone resorption follows tooth extraction. Immediate implant placement alone is not sufficient to prevent this phenomenon. CASE PRESENTATION: In this clinical case, a fractured upper first premolar was replaced by an immediate implant and a customized healing abutment designed on the perimeter of the extractive alveolus. After 3 months, the implant was restored. The facial and interdental  soft  tissue  was maintained with  appreciable  success after 5 years. The pre- and 5-year-post-treatment CT scans showed no bone loss. CONCLUSION: The use of an interim customized healing abutment helps preventing hard and soft tissues collapse.  This technique is very straightforward and might represent a smart preservation strategy when there is no indication for adjunctive hard or soft tissue grafting.

Extending the allelic spectrum at noncoding risk loci of orofacial clefting.

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Endocytosis as a stabilizing mechanism for tissue homeostasis.

Cells in tissues communicate by secreted growth factors (GF) and other signals. An important function of cell circuits is tissue homeostasis: maintaining proper balance between the amounts of different cell types. Homeostasis requires negative feedback on the GFs, to avoid a runaway situation in which cells stimulate each other and grow without control. Feedback can be obtained in at least two ways: endocytosis in which a cell removes its cognate GF by internalization and cross-inhibition in which a GF down-regulates the production of another GF. Here we ask whether there are design principles for cell circuits to achieve tissue homeostasis. We develop an analytically solvable framework for circuits with multiple cell types and find that feedback by endocytosis is far more robust to parameter variation and has faster responses than cross-inhibition. Endocytosis, which is found ubiquitously across tissues, can even provide homeostasis to three and four communicating cell types. These design principles form a conceptual basis for how tissues maintain a healthy balance of cell types and how balance may be disrupted in diseases such as degeneration and fibrosis.

Peri-implant health.

OBJECTIVE: The aim is to define clinical and histologic characteristics of peri-implant tissues in health and describe the mucosa-implant interface. IMPORTANCE: An understanding of the characteristics of healthy peri-implant tissues facilitates the recognition of disease (i.e., departure from health). FINDINGS: The healthy peri-implant mucosa is, at the microscopic level, comprised of a core of connective tissue covered by either a keratinized (masticatory mucosa) or non-keratinized epithelium (lining mucosa). The peri-implant mucosa averages about 3 to 4 mm high, and presents with an epithelium (about 2 mm long) facing the implant surface. Small clusters of inflammatory cells are usually present in the connective tissue lateral to the barrier epithelium. Most of the intrabony part of the implant appears to be in contact with mineralized bone (about 60%), while the remaining portion faces bone marrow, vascular structures, or fibrous tissue. During healing following implant installation, bone modeling occurs that may result in some reduction of the marginal bone level. CONCLUSIONS: The characteristics of the peri-implant tissues in health are properly identified in the literature, including tissue dimensions and composition. Deviation from the features of health may be used by the clinician (and researcher) to identify disease, including peri-implant mucositis and peri-implantitis.

Influence of bisphosphonates on the adherence and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces.

OBJECTIVES: To evaluate the effects of sodium alendronate (SA) and zoledronic acid (ZA), on the adhesion and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces considering cell functions related to an effective mucosal barrier around the implant. MATERIALS AND METHODS: Cells were seeded onto titanium discs and incubated for 24 h. Then, serum-free DMEM containing selected bisphosphonates (0, 0.5, 1, or 5 µM) was added for 24 and 48 h. Factors related to the achievement of an effective mechanical and immunological barrier-cell adhesion, viability, collagen epidermal growth factor, and immunoglobulin synthesis-were evaluated. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests as well as by ANOVA and Tukey's tests, (α = 0.05). RESULTS: The presence of bisphosphonates culminated in lower cell adhesion to the titanium discs, particularly for SA at 5 µM (40%) and ZA at all concentrations (from 30 to 50%, according to increased concentrations). Reduced cell viability occurred after exposing these cells to ZA (40%); however, only 5 µM SA-treated cells had decreased viability (30%). Reduced synthesis of growth factors and collagen was observed when cells were reated with ZA (20 and 40%, respectively), while about 70% of IgG synthesis was enhanced. CONCLUSION: Bisphosphonates negatively affected the adhesion and metabolism of oral mucosal cells, and this effect was related to the type of bisphosphonate as well as to concentration and period of treatment. CLINICAL RELEVANCE: The negative effects of bisphosphonates on oral mucosal cells can hamper the formation of an effective biological seal in osseointegrated implants.

Development and analysis of scaffold-free adipose spheroids.

Adipose tissue plays a crucial role in metabolic syndrome, autoimmune diseases, and many cancers. Because of adipose's role in so many aspects of human health, there is a critical need for in vitro models that replicate adipose architecture and function. Traditional monolayer models, despite their convenience, are limited, showing heterogeneity and functional differences compared to 3D models. While monolayer cultures struggle with detachment and inefficient differentiation, healthy adipocytes in 3D culture accumulate large lipid droplets, secrete adiponectin, and produce low levels of inflammatory cytokines. The shift from monolayer models to more complex 3D models aims to better replicate the physiology of healthy adipose tissue in culture. This study introduces a simple and accessible protocol for generating adipose organoids using a scaffold-free spheroid model. The method, utilizing either 96-well spheroid plates or agarose micromolds, demonstrates increased throughput, uniformity, and ease of handling compared to previous techniques. This protocol allows for diverse applications, including drug testing, toxin screening, tissue engineering, and co-culturing. The choice between the two methods depends on the experimental goals, with the 96-well plate providing individualized control and the micromold offering scale advantages. The outlined protocol covers isolation, expansion, and characterization of stromal vascular fraction cells, followed by detailed steps for spheroid formation and optional downstream analyses.

Regional adiposity and insulin sensitivity - interactions with menopause and HIV in middle-aged Black African women.

OBJECTIVE: To explore depot-specific functional aspects of adipose tissue, examining the putative role for menopause and HIV status on insulin sensitivity (SI) and beta-cell function in Black South African women. METHODS: Women (n = 92) from the Middle-Aged Soweto Cohort, including premenopausal HIV-negative (n = 21); premenopausal women living with HIV (WLWH; n = 11); postmenopausal HIV-negative (n = 42); postmenopausal WLWH (n = 18) underwent the following tests: body composition (dual energy x-ray absorptiometry); fasting bloods for sex hormones, inflammation and adipokines; frequently sampled intravenous glucose tolerance test for SI and beta-cell function (disposition index, DI); abdominal (aSAT) and gluteal subcutaneous adipose tissue (gSAT) biopsies for cell size and mRNA expression of adipokines, inflammation, and estrogen receptors [ER]. RESULTS: Depot-specific associations between gene expression and insulin parameters did not differ by HIV or menopause status. Pooled analysis showed significant models for SI (P = 0.002) and DI (P = 0.003). Higher SI was associated with lower leptin and CD11c expression in aSAT and higher adiponectin in gSAT. Higher DI was associated with higher aSAT and gSAT expression of adiponectin, LPL, ERα, and PPARγ, and lower leptin in aSAT. WLWH had higher expression of adiponectin and lower expression of leptin in both aSAT (P = 0.002 and P = 0.005) and gSAT (P = 0.004 and P = 0.002), respectively, and a larger proportion of smaller cells in aSAT (P < 0.001). CONCLUSION: Insulin sensitivity and beta cell function were distinctively associated with aSAT and gSAT. While menopause did not influence these relationships, HIV had a significant effect on adipose tissue, characterised by variations in cell size distribution and transcript levels within the depots.

Listening in on Multicellular Communication in Human Tissue Immunology.

Immune responses in human tissues rely on the concerted action of different cell types. Inter-cellular communication shapes both the function of the multicellular interaction networks and the fate of the individual cells that comprise them. With the advent of new methods to profile and experimentally perturb primary human tissues, we are now in a position to systematically identify and mechanistically dissect these cell-cell interactions and their modulators. Here, we introduce the concept of multicellular hubs, functional modules of immune responses in tissues. We outline a roadmap to discover multicellular hubs in human tissues and discuss how emerging technologies may further accelerate progress in this field.

Systemic and Adipose Tissue Redox Status in Sprague-Dawley Rats Fed Normal- and High-Fat Diets Supplemented with Lycopene.

Dietary patterns high in fat influence local and systemic oxidative stress through adipose tissue (AT) accrual and increased reactive oxygen species generation. Lycopene, a carotenoid with antioxidant functionality, may mitigate excess oxidative stress, yet the lipophilic nature of this compound may limit its functionality if sequestered by AT. Thus, it is critical to elucidate whether lycopene's efficacy is limited based on adiposity. The purpose of this study was to investigate the influence of lycopene-supplemented normal- and high-fat diets on systemic and AT redox status. Male Sprague-Dawley rats (n = 18) were fed a 30% normal-fat (NFD) or 60% high-fat (HFD) purified diet supplemented with 100 mg of lycopene/day. Body weight and visceral AT mass, as well as serum and AT lycopene, lipid peroxides, and antioxidant capacity (AC), were assessed after 3, 7, and 10 weeks of supplementation. At week 10, AT mass was significantly higher (P = .028) in the HFD group, yet there were no significant differences in serum or AT lycopene concentrations or lipid peroxides between groups. Additionally, AT in the HFD group exhibited significantly greater lipophilic AC (27.6% higher, P = .031). Results suggest that excess adiposity did not negatively influence circulating lycopene, nor did it limit its antioxidant functionality.

Bridging scales: From cell biology to physiology using in situ single-cell technologies.

Biological organization crosses multiple spatial scales: from molecular, cellular, to tissues and organs. The proliferation of molecular profiling technologies enables increasingly detailed cataloging of the components at each scale. However, the scarcity of spatial profiling has made it challenging to bridge across these scales. Emerging technologies based on highly multiplexed in situ profiling are paving the way to study the spatial organization of cells and tissues in greater detail. These new technologies provide the data needed to cross the scale from cell biology to physiology and identify the fundamental principles that govern tissue organization. Here, we provide an overview of these key technologies and discuss the current and future insights these powerful techniques enable.

Sphingolipid Profiling Reveals Different Extent of Ceramide Accumulation in Bovine Retroperitoneal and Subcutaneous Adipose Tissues.

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT.

AT1 receptor antagonism promotes bone loss attenuation in experimental periodontitis, blocks inflammatory mediators, and upregulates antioxidant enzymes and bone formation markers.

BACKGROUND: The initiation and progression of periodontitis might involve a local renin-angiotensin system in periodontal tissue. This study hypothesized that Losartan treatment could promote protection to rats submitted to experimental periodontitis (EP) by attenuating alveolar bone loss due to reduction in inflammatory cytokines, better reactive oxidant species regulation and maintenance of the balance between bone formation and resorption factors. METHODS: One hundred and thirty rats were submitted to EP with a silk suture thread (4.0) placed around the lower right first molar for 1, 3, 7, and 14 consecutive days. The study comprised four groups: G1-control without EP; G2-animals with EP treated with water; G3-Losartan-treated animals (treatment started at the same day of EP induction), and G4-animals previously treated with Losartan for 30 days followed by induction of EP and continuity of treatment. RESULTS: G2 rats had greater bone loss volume, increased number, and thickness and decreased separation of trabeculae. On the other hand, G4 animals showed significant improvements in these parameters. Histological analysis revealed that EP favors inflammatory cell infiltration and junctional epithelium, cementum with alveolar bone crest destruction, but animals pretreated with Losartan (G4) did not show these features. Although the G3 animals did not demonstrate the improvements detected in G4, mRNA expression results were similar. In mandibular tissue, EP promoted mRNA increases for ACE, AT1 receptor, and inflammatory mediators as well as decreases for antioxidant enzymes. However, Losartan treatments attenuated these responses in addition to promoting an increase in bone formation markers and transcription factors. CONCLUSION: AT1 receptor modulates EP progression.

Volumetric evaluation of lumbar epidural fat distribution in epidural lipomatosis and back pain in patients who are obese: introducing a novel technique (Fat Finder algorithm).

Objective: Spinal epidural lipomatosis (EL) represents an excessive deposition of unencapsulated adipose tissue in the spinal canal that can result in chronic back pain in patients who are obese with and without diabetes. We aim to calculate the total volumetric epidural fat on lumbar spine MRI in a predominately obese population and correlate total epidural fat to lower back pain (LBP) and body mass index (BMI). Research design and methods: We developed a program (Fat Finder) to quantify volumetric distribution of epidural fat throughout the lumbar spine. Eleven patients with LBP were imaged using two MRI protocols: parallel axial slices and conventional clinical protocol. The distribution of epidural fat per level was analyzed and normalized to the spinal canal size. Results: Our sample had an average age of 59.9 years and BMI of 31.57 kg/m2. EL subgroup consisted of seven patients. The L2-L5 total fat volume was 3477.6 mm3 (1431.1-5595.9) in the EL group versus 1783.8 mm3 (815.0-2717.5) in the age-similar non-EL group. A higher percentage of fat volume in the canal was associated with higher LBP scores. The fat percentage was 32.2% among patients with EL versus 15.4% for age-similar non-EL with LBP score of 6.1 and 4.0, respectively. Conclusions: The Fat Finder is a novel volumetric method to quantify epidural lumbar spinal fat. The epidural fat favors the lower spinal segment with direct proportionality between the fat volume and LBP score, independent of BMI.

Continuum of Gene-Expression Profiles Provides Spatial Division of Labor within a Differentiated Cell Type.

Single-cell gene expression reveals the diversity within a differentiated cell type. Often, cells of the same type show a continuum of gene-expression patterns. The origin of such continuum gene-expression patterns is unclear. To address this, we develop a theory to understand how a continuum provides division of labor in a tissue in which cells collectively contribute to several tasks. We find that a continuum is optimal when there are spatial gradients in the tissue that affect the performance in each task. The continuum is bounded inside a polyhedron whose vertices are expression profiles optimal at each task. We test this using single-cell gene expression for intestinal villi and liver hepatocytes, which form a curved 1D trajectory and a full 3D tetrahedron in gene-expression space, respectively. We infer the tasks for both cell types and characterize the spatial zonation of the task-specialist cells. This approach can be generally applied to other tissues.

Peri-implant health.

OBJECTIVE: The aim is to define clinical and histologic characteristics of peri-implant tissues in health and describe the mucosa-implant interface. IMPORTANCE: An understanding of the characteristics of healthy peri-implant tissues facilitates the recognition of disease (i.e., departure from health). FINDINGS: The healthy peri-implant mucosa is, at the microscopic level, comprised of a core of connective tissue covered by either a keratinized (masticatory mucosa) or non-keratinized epithelium (lining mucosa). The peri-implant mucosa averages about 3 to 4 mm high, and presents with an epithelium (about 2 mm long) facing the implant surface. Small clusters of inflammatory cells are usually present in the connective tissue lateral to the barrier epithelium. Most of the intrabony part of the implant appears to be in contact with mineralized bone (about 60%), while the remaining portion faces bone marrow, vascular structures, or fibrous tissue. During healing following implant installation, bone modeling occurs that may result in some reduction of the marginal bone level. CONCLUSIONS: The characteristics of the peri-implant tissues in health are properly identified in the literature, including tissue dimensions and composition. Deviation from the features of health may be used by the clinician (and researcher) to identify disease, including peri-implant mucositis and peri-implantitis.

Flow Cytometry and Lineage Tracing Study for Identification of Adipocyte Precursor Cell (APC) Populations.

Flow cytometry and fluorescence-activated cell sorting (FACS) techniques have significantly advanced the characterization of adipocyte precursor cell (APC) populations. They allow immunophenotyping, quantification, and isolation of distinct populations, which is critical for understanding adipose tissue development and homeostasis. Here, we describe the identification and purification of adipocyte precursor cells using flow cytometry and FACS, defined by previously established surface marker profiles. In addition, we describe the mouse models and whole adipose tissue visualization techniques that will enable us to characterize the plasticity and the cellular origin of APCs.