Harnessing urban analytics and machine learning for sustainable urban development: A multidimensional framework for modeling environmental impacts of urbanization in Saudi Arabia.

Sustainable urban development is crucial for managing natural resources and mitigating environmental impacts induced by rapid urbanization. This study demonstrates an integrated framework using machine learning-based urban analytics techniques to evaluate spatiotemporal urban expansion in Saudi Arabia (1987-2022) and quantify impacts on leading land, water, and air-related environmental parameters (EPs). Remote sensing and statistical techniques were applied to estimate vegetation health, built-up area, impervious surface, water bodies, soil characteristics, thermal comfort, air pollutants (PM2.5, CH4, CO, NO2, SO2), and nighttime light EPs. Regression assessment and Principal Component Analysis (PCA) were applied to assess the relationships between urban expansion and EPs. The findings highlight the substantial growth of urban areas (0.067%-0.14%), a decline in soil moisture (16%-14%), water bodies (60%-22%), a nationwide increase of PM2.5 (44 µg/m3 to 73 µg/m3) and night light intensity (0.166-9.670) concentrations resulting in significant impacts on land, water, and air quality parameters. PCA showed vegetation cover, soil moisture, thermal comfort, PM2.5, and NO2 are highly impacted by urban expansion compared to other EPs. The results highlight the need for effective and sustainable interventions to mitigate environmental impacts using green innovations and urban development by applying mixed-use development, green space preservation, green building technologies, and implementing renewable energy approaches. The framework recommended for environmental management in this study provides a robust foundation for evidence-based policies and adaptive management practices that balance economic progress and environmental sustainability. It will also help policymakers and urban planners in making informed decisions and promoting resilient urban growth.

Decoding seasonal variability of air pollutants with climate factors: A geostatistical approach using multimodal regression models for informed climate change mitigation.

In response to changes in climatic patterns, a profound comprehension of air pollutants (AP) variability is vital for enhancing climate models and facilitating informed decision-making in nations susceptible to climate change. Earlier research primarily depended on limited models, potentially neglecting intricate relationships and not fully encapsulating associations. This study, in contrast, probed the spatiotemporal variability of airborne particles (CO, CH4, SO2, and NO2) under varying climatic conditions within a climate-sensitive nation, utilizing multiple regression models. Spatial and seasonal AP data were acquired via the Google Earth Engine platform, which indicated elevated AP concentrations in primarily urban areas. Remarkably, the average airborne particle levels were lower in 2020 than in 2019, though they escalated during winter. The study employed linear regression, Pearson's correlation (PC), Spearman rank correlation models, and Geographically Weighted Regression (GWR) models to probe the relationship between pollutant variability and climatic elements such as rainfall, temperature, and humidity. Across all seasons, APs showed a negative correlation with rainfall while displaying positive correlations with temperature and humidity. The GWR and PC models produced the most reliable results from all the models employed, with the GWR model superseding the rest. Moreover, heightened aerosol levels were detected within a rainfall range of 600 mm/season, a temperature range of 25-30 °C, and humidity levels of 75 %-85 %. Overall, this study emphasizes the growing levels of APs in correlation with meteorological changes. By adopting a comprehensive approach and considering multiple factors, this research provides a more sophisticated understanding of the relationship between AP variability and climatic shifts.

Spatiotemporal characterization of relative humidity trends and influence of climatic factors in Bangladesh.

The frequency and intensity of climate change and resulting impacts are more prevalent in South Asian countries, particularly in Bangladesh. Relative humidity (RH) is a crucial aspect of climate, and higher RH variability has far-reaching impacts on human health, agriculture, environment, and infrastructure. While temperature and rainfall have gained much research attention, RH studies have received scant attention in the research literature. This study investigated the trends and variability of RH levels in Bangladesh and the influence of other meteorological factors over the past 40 years. Variabilities in the meteorological factors were identified by calculating descriptive statistics. Innovative trend analysis (ITA) and Mann-Kendall test (MK-test) methods were utilized to assess monthly, seasonal, and annual trends. The magnitude of temperature, rainfall, and windspeed influences on RH variability were identified using Pearson's correlation, Spearman rank correlation, and Kendall correlation model. Variability analysis showed higher spatial variations in RH levels across the country, and RH skewed negatively in all stations. Results reveal that daily, monthly, seasonal, and annual trends of RH exhibited positive trends in all stations, with an increasing rate of 0.083-0.53% per year in summer, 0.43-0.68% per year in winter, and 0.58-0.31% per year in the rainy season. Both ITA and MK-test provided consistent results, indicating no discrepancies in trend results. All three models indicate that temperature, rainfall, and windspeed have weak to moderate positive influences on changing RH levels in Bangladesh. The study will contribute to decision-making to improve crop yields, health outcomes, and infrastructure efficiency.

The role of the microbiome on fish mucosal immunity under changing environments.

The environment is crucial for fish as their mucosal surfaces face continuous challenges in the water. Fish mucosal surfaces harbor the microbiome and mucosal immunity. Changes in the environment could affect the microbiome, thus altering mucosal immunity. Homeostasis between the microbiome and mucosal immunity is crucial for the overall health of fish. To date, very few studies have investigated mucosal immunity and its interaction with the microbiome in response to environmental changes. Based on the existing studies, we can infer that environmental factors can modulate the microbiome and mucosal immunity. However, we need to retrospectively examine the existing literature to investigate the possible interaction between the microbiome and mucosal immunity under specific environmental conditions. In this review, we summarize the existing literature on the effects of environmental changes on the fish microbiome and mucosal immunity. This review mainly focuses on temperature, salinity, dissolved oxygen, pH, and photoperiod. We also point out a gap in the literature and provide directions to go further in this research field. In-depth knowledge about mucosal immunity-microbiome interaction will also improve aquaculture practices by reducing loss during environmental stressful conditions.

Freshwater transfer affected intestinal microbiota with correlation to cytokine gene expression in Asian sea bass.

As a catadromous fish, Asian sea bass (Lates calcarifer) juveniles migrate from seawater (SW) to freshwater (FW) for growth and development. During migration, they undergo physiological changes to acclimate to environmental salinity. Thus, it is crucial to understand how SW-to-FW migration affects the gut microbiota of catadromous fish. To the best of our knowledge, no study has revealed the effects of transfer to hypotonic environments on a catadromous fish microbiota. In this study, we aimed to determine the effects of FW transfer on the microbiota and cytokine gene expression in the intestines of juvenile catadromous Asian sea bass. The relationship between the water and the gut microbiota of this euryhaline species was also examined. We found that FW transfer affected both mucosa- and digesta-associated microbiota of Asian sea bass. Plesiomonas and Cetobacterium were dominant in both the mucosa- and digesta-associated microbiota of FW-acclimated sea bass. The pathogenic genera Vibrio, Staphylococcus, and Acinetobacter were dominant in the SW group. Although dominant fish microbes were present in the water, fish had their own unique microbes. Vitamin B6 metabolism was highly expressed in the FW fish microbiota, whereas arginine, proline, and lipid metabolism were highly expressed in the SW fish microbiota. Additionally, the correlation between cytokine gene expression and microbiota was found to be affected by FW transfer. Taken together, our results demonstrated that FW transfer altered the composition and functions of mucosa- and digesta-associated microbiota of catadromous Asian sea bass intestines, which correlated with cytokine gene expression.

Thyroid Stem Cell Speciation-a Major Role for PKC.

Instructive signals that delineate the formation of thyroid follicles by thyrotropin (TSH) in stem cells are complex. Here, we have examined the role of protein kinase C (PKC) by using a unique Gαq/11 biased small molecule (MSq1) to develop thyroid progenitor cells. Mouse embryonic stem cells (mESCs) were differentiated into anterior endoderm cells and treated with either TSH or MSq1 in the presence or absence of PKC inhibitors. The transcriptional and translational response of key thyroid markers-sodium iodide symporter (NIS), thyroglobulin (TG), and thyrotropin receptor (TSHR) as well as potential signaling molecules-were then analyzed. The data confirmed that MSq1 is a potent Gαq/11 activator with a major increase in Gαq/11 signaling when compared to TSH. MSq1 activation resulted in an increase in thyroid-specific genes, demonstrating that enhanced PKC signaling was able to induce their expression. The specificity of the PKC signals over the protein kinase A (PKA) pathway in regulating thyroid gene expression was shown by using a specific PKC enzyme inhibitor. The data revealed that TG and NIS expression were suppressed in the presence of the PKC inhibition but, in contrast, were not influenced by PKA inhibition. This indicated that PKC activation was the dominant pathway in the inductive process for thyroid hormone production. Furthermore, by examining PKC isoforms we found that PKCξ was the predominant form in the ES cells that mediated the effects. Since PKCξ can lead to activation of transforming growth factor-ß-activated kinase (pTAK1), and its downstream effector nuclear factor κB (NFκB) complex, this demonstrated the involvement of the TAK1/NFκB pathway in thyroid speciation.

Signal responses to neutral TSH receptor antibody - A cycle of damage in the pathophysiology of Graves' disease.

BACKGROUND: Graves' disease is associated with TSH receptor (TSHR) antibodies of variable bioactivity including "neutral" antibodies (N-TSHR-Ab) that bind to the hinge region of the TSHR ectodomain. We have previously found that such antibodies induced thyroid cell apoptosis via excessive mitochondrial and ER stress with elevated reactive oxygen species (ROS). However, the detailed mechanisms by which excess ROS was induced remained unclear. OBJECTIVES: To determine how ROS is induced by N-TSHR-monoclonal antibodies (mAb, MC1) mediated signaling and to measure stress in polyorganelles. METHODS: Total ROS and mitochondrial ROS was measured by fluorometry of live rat thyrocytes. Live-cell imaging of labelled organelles was carried out using red or green fluorescent dyes. Proteins were detected by Li-Cor Western immunoblots and immunocytochemistry. RESULTS: Endocytosis of N-TSHR-mAb induced ROS, disturbed vesicular trafficking, damaged organelles and failed to induce lysosomal degradation and autophagy. We found that the endocytosis triggered signaling cascades involving Gα13 and PKC-δ leading to intrinsic thyroid cell apoptosis. CONCLUSIONS: These studies define the mechanism of ROS induction in thyroid cells following the endocytosis of N-TSHR-Ab/TSHR complexes. We suggest that a viscous cycle of stress initiated by cellular ROS and induced by N-TSHR-mAbs may orchestrate overt intra-thyroidal, retro-orbital, and intra-dermal inflammatory autoimmune reactions in patients with Graves' disease.

A GIS and remote sensing approach for measuring summer-winter variation of land use and land cover indices and surface temperature in Dhaka district, Bangladesh.

Rapid urbanization has induced land use and land cover change (LULC) that increases land surface temperature (LST). Analyzing seasonal variations of LULC and LST is a precondition for mitigating heat island effects and promoting a sustainable living environment. The objective of this study is to explore the association between the seasonal LST dynamics and LULC indices for the Dhaka district of Bangladesh. The LULC indices are comprised of the Normalized Difference Vegetation Index (NDVI), Normalized Difference Built-up Index (NDBI), Normalized Difference Bareness Index (NDBAI), and Modified Normalized Difference Water Index (MNDWI). The results show that the LULC effect on LST in Dhaka is significant, with an increase in summer season LST from 34.58 °C to 37.66 °C and in winter season LST from 24.710C to 26.24 °C. Predictably, the highest and lowest LST values were observed in the built-up and vegetation-covered areas, respectively. Secondly, the correlation values indicate a significant inverse correlation (R2 > 0.50) between NDVI and LST, as well as MNDWI and LST. On the contrary, positive correlations were observed between NDBI and LST, and between NDBAI and LST for both the summer and winter seasons. Finally, subsequent vegetation decline (-69.34%) and increasing built-up area (+11.30%) between 2000 and 2020 in Dhaka district were found to be the most significant factors for the increasing trend and spatial heterogeneity of LST in Dhaka. The methodological approach of this study offers a low-cost efficient technique for monitoring LST hotspots, which can guide land use planners and urban managers for spatial intervention to ensure a livable environment.

Brief Report - Monoclonal Antibodies Illustrate the Difficulties in Measuring Blocking TSH Receptor Antibodies.

TSH receptor (TSHR) antibodies are the cause of Graves' disease and may also be found in patients with Hashimoto's thyroiditis. They come in at least three varieties: thyroid stimulating, thyroid blocking and neutral. The measurement of TSH receptor antibodies in Graves' disease and Hashimoto's thyroiditis is a common clinical activity and can be useful in diagnosis and prognosis. We show that it is not possible to detect the blocking variety of TSHR antibody in patients with Graves' disease because the stimulating antibody may overwhelm the measurement of blocking in the bioassays available for their measurement and may blind the valid interpretation of the results. To help explain this in more detail we show a series of studies with monoclonal TSHR antibodies which support this conclusion.

Environmental benefits of blue ecosystem services and residents' willingness to pay in Khulna city, Bangladesh.

Nature-based solutions for urban problems gaining popularity globally. The well-functioning ecosystem could offer a nature-based solution to many urban problems including water, drainage and flooding problems. Therefore, conservation and restoration of urban blue ecosystem components such as pond scape are crucial. This research taking Khulna city of Bangladesh as a case has examined the low-income fringe community's willingness to pay (WTP) for conservation and restoration of pond scape/blue ecosystem service (BES) in their locality from where they benefit. The various types of ecosystem services enjoyed by the local community were identified. To assess the community's WTP for conservation and restoration of pond scape, the payment card approach of the Contingent Valuation Method (CVM) was used. Three environmental attributes were considered to assess the existing condition of the blue ecosystem services in the study area. Findings show that 54% of respondents are not satisfied with the existing conditions of the ecosystem services resulting from the pond scape. Respondent's WTP for eleven types of service facilities was calculated. Results show that only 65.20% are eager to pay an amount of 38 Tk to 138 Tk per month for different service facilities. It means about one-third of the community people want to be free riders. The influences of different attributes of the respondents on their WTP were also analyzed. Education, income, and house-ownership appear to have a positive significant influence on WTP for conservation and restoration of pond scape in the study area. In line with the findings if policy measures are taken without further delay it would help conserve the remaining pond scape.

Mechanisms in Graves Eye Disease: Apoptosis as the End Point of Insulin-Like Growth Factor 1 Receptor Inhibition.

Background: Graves' eye disease, also called Graves' orbitopathy (GO), is a potentially debilitating autoimmune disease associated with retro-orbital inflammation and tissue expansion, involving both fibroblasts and adipocytes, resulting in periorbital edema, worsening proptosis, and muscle dysfunction with diplopia and may ultimately threaten sight. Accumulating evidence has indicated that autoantibodies to the thyrotropin receptor (TSHR), which induce the hyperthyroidism of Graves' disease, also help mediate the pathogenesis of the eye disease in susceptible individuals through TSHR expression on retro-orbital cells. Since it has long been known that the effects of insulin-like growth factor 1 (IGF-1) and thyrotropin are additive, recent clinical trials with a human monoclonal IGF-1 receptor blocking antibody (teprotumumab; IGF-1R-B-monoclonal antibody [mAb]) have demonstrated its ability to induce significant reductions in proptosis, diplopia, and clinical activity scores in patients with GO. However, the molecular mechanisms by which such an antibody achieves this result is unclear. Methods: We have used Li-Cor In-Cell Western, Western blot, and immunohistochemistry to define levels of different proteins in mouse and human fibroblast cells. Proteomic array was also used to define pathway signaling molecules. Using CCK-8 and BrdU cell proliferation ELISA, we have analyzed proliferative response of these cells to different antibodies. Results: We now show that a stimulating TSHR antibody was able to induce phosphorylation of the IGF-1R and initiate both TSHR and IGF-1R signaling in mouse and human fibroblasts. IGF-1R-B-mAb (1H7) inhibited all major IGF-1R signaling cascades and also reduced TSHR signaling. This resulted in the antibody-induced suppression of autophagy as shown by inhibition of multiple autophagy-related proteins (Beclin1, LC3a, LC3b, p62, and ULK1) and the induction of cell death by apoptosis as evidenced by activation of cleaved caspase 3, FADD, and caspase 8. Furthermore, this IGF-1R-blocking mAb suppressed serum-induced perkin and pink mitophagic proteins. Conclusions: Our observations clearly indicated that stimulating TSHR antibodies were able to enhance IGF-1R activity and contribute to retro-orbital cellular proliferation and inflammation. In contrast, an IGF-1R-B-mAb was capable of suppressing IGF-1R signaling leading to retro-orbital fibroblast/adipocyte death through the cell-extrinsic pathway of apoptosis. This is likely the major mechanism involved in proptosis reduction in patients with Graves' eye disease treated by IGF-1R inhibition.

Rescue of thyroid cells from antibody induced cell death via induction of autophagy.

BACKGROUND: Graves' disease (GD) is associated with thyroid stimulating hormone (TSH) receptor (TSHR) antibodies of variable bioactivity. We have previously characterized "neutral" TSHR antibodies (N-TSHR-Abs) that bind to the hinge region of the TSHR ectodomain. We showed that an N-TSHR monoclonal antibody (mAb) failed to induce any G proteins to sustain survival signaling and lead to excessive stress and apoptosis. Furthermore, the addition of TSH, or the antioxidant N-acetyl-l-cysteine (NAC), rescued N-TSHR-mAb-induced apoptotic death. However, the detailed mechanisms of this rescue remained unclear. METHODS: Autophagy is activated in response to diverse stress related stimuli so we have, therefore, studied the autophagy response in rat thyroid cells (FRTL-5) during N-TSHR-mAb induced thyrocyte stress and apoptosis using the In Cell Western technique for quantitation along with immunocytochemistry. RESULTS: Under starvation conditions with N-TSHR-mAb the addition of TSH or NAC prevented thyroid cell death by enhancing autophagy. This was evidenced by elevated levels of autophagy related proteins including beclin 1, LC3A, LC3B, ULK1, p62, and also activated pink and perkin mitophagy related proteins. The phenomenon was further confirmed by image analyses using Cyto-ID and Mito-ID autophagy detection systems. We also found that either TSH or NAC enhanced PKA, Akt, mTORC, AMPK, Sirtuins, PGC1α, NRF-2, mitofusin-2, TFAM and catalase in the N-TSHR-mAb stressed cells. Thus TSH or NAC restored cell survival signaling which reduced cell stress and enhanced mitochondrial biogenesis. The N-TSHR-mAb also activated cytochrome-C, Bax, caspase-9, caspase-3A, and had less effect on FADD or caspase-8 indicating activation of the intrinsic pathway for apoptosis. CONCLUSIONS: These findings indicated that TSH or antioxidant can rescue thyroid cells from N-TSHR-mAb induced apoptosis via enhanced autophagy. These observations signify that N-TSHR-mAb in GD under low TSH conditions caused by the hyperthyroidism could be detrimental for thyrocyte survival which would be another factor able to precipitate ongoing autoinflammation.

Long Term Rescue of the TSH Receptor Knock-Out Mouse - Thyroid Stem Cell Transplantation Restores Thyroid Function.

The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500µU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.

Derivation and 97% Purification of Human Thyroid Cells From Dermal Fibroblasts.

Background: The success in rescuing thyroid deficiency in mice using thyroid cells derived from embryonic stem (ES) cells, together with the discovery of human induced pluripotent stem cells (iPSCs) from somatic cells, has raised the possibility of patient-specific thyroid cell replacement. In this study we demonstrate that human thyroid follicular cells can be derived from human iPSCs and show the ability of highly purified and differentiated cells to secrete thyroid hormone. Research Design and Methods: Human iPSCs were derived from adult skin fibroblasts using RNA reprogramming and differentiated in vitro into thyroid follicular cells by exposure to activin A, ethacridine and TSH as we have previously described for human ES cells. The resulting thyroid cells were then highly purified using double antibody cell sorting. Results: The iPSCs derived from human dermal fibroblasts showed stem cell-like morphologic changes and expressed pluripotent stem cell markers as assessed using qPCR, immunofluorescence staining, and FACS analysis. These cells retained their pluripotential characteristics as shown by teratoma formation after murine transplantation. Definitive endoderm cells were induced with activin A and the transcription factor TAZ was significantly induced on ethacridine treatment and translocated to the nucleus. Thyroid transcription factors NKX2-1 and PAX8 were also highly expressed in activin A derived endoderm cells and further induced by ethacridine. Following terminal differentiation with TSH, there was enhanced thyroid follicle formation, high expression of the thyroid specific genes-TG, TPO, TSHR and NIS, and secretion of thyroid hormone (T4) in vitro. Furthermore, we were able to achieve a 97% purification of TSHR+/NIS+ expressing cells after differentiation using a single purification procedure. Conclusions: These findings demonstrate that mature adult dermal fibroblasts can be matured into human iPSCs which have the potential to form functional thyroid follicular cells. This lays the groundwork for future person-specific thyroid regenerative therapy.

A Gq Biased Small Molecule Active at the TSH Receptor.

G protein coupled receptors (GPCRs) can lead to G protein and non-G protein initiated signals. By virtue of its structural property, the TSH receptor (TSHR) has a unique ability to engage different G proteins making it highly amenable to selective signaling. In this study, we describe the identification and characterization of a novel small molecule agonist to the TSHR which induces primary engagement with Gαq/11. To identify allosteric modulators inducing selective signaling of the TSHR we used a transcriptional-based luciferase assay system with CHO-TSHR cells stably expressing response elements (CRE, NFAT, SRF, or SRE) that were capable of measuring signals emanating from the coupling of Gαs , Gαq/11, Gßγ, and Gα12/13, respectively. Using this system, TSH activated Gαs , Gαq/11, and Gα12/13 but not Gßγ. On screening a library of 50K molecules at 0.1,1.0 and 10 µM, we identified a novel Gq/11 agonist (named MSq1) which activated Gq/11 mediated NFAT-luciferase >4 fold above baseline and had an EC50= 8.3 × 10-9 M with only minor induction of Gαs and cAMP. Furthermore, MSq1 is chemically and structurally distinct from any of the previously reported TSHR agonist molecules. Docking studies using a TSHR transmembrane domain (TMD) model indicated that MSq1 had contact points on helices H1, H2, H3, and H7 in the hydrophobic pocket of the TMD and also with the extracellular loops. On co-treatment with TSH, MSq1 suppressed TSH-induced proliferation of thyrocytes in a dose-dependent manner but lacked the intrinsic ability to influence basal thyrocyte proliferation. This unexpected inhibitory property of MSq1 could be blocked in the presence of a PKC inhibitor resulting in derepressing TSH induced protein kinase A (PKA) signals and resulting in the induction of proliferation. Thus, the inhibitory effect of MSq1 on proliferation resided in its capacity to overtly activate protein kinase C (PKC) which in turn suppressed the proliferative signal induced by activation of the predomiant cAMP-PKA pathway of the TSHR. Treatment of rat thyroid cells (FRTL5) with MSq1 did not show any upregulation of gene expression of the key thyroid specific markers such as thyroglobulin(Tg), thyroid peroxidase (Tpo), sodium iodide symporter (Nis), and the TSH receptor (Tshr) further suggesting lack of involvement of MSq1 and Gαq/11 activation with cellular differentation. In summary, we identified and characterized a novel Gαq/11 agonist molecule acting at the TSHR and which showed a marked anti-proliferative ability. Hence, Gq biased activation of the TSHR is capable of ameliorating the proliferative signals from its orthosteric ligand and may offer a therapeutic option for thyroid growth modulation.

Epigenetic Changes During Human Thyroid Cell Differentiation.

Objective: It has been demonstrated that the transcription factors TAZ (transcriptional coactivator with PDZ-binding motif), paired box gene 8 (PAX8), and NK2 homeobox 1 (NKX2-1) are coexpressed in the nucleus of thyroid cells. Furthermore, TAZ is known to enhance the transcriptional activity of PAX8 and NKX2-1 as well as the key thyroid-specific gene, thyroglobulin (TG), suggesting a critical role for TAZ in the control of thyroid cell speciation. We previously reported that the small molecule ethacridine, identified as a TAZ activator, was able to induce thyroid-specific transcription in endodermal cells differentiated from human embryonic stem (hES) cells using activin A. Since transcription factors are epigenetically regulated in cell differentiation, we investigated the epigenetic changes in the promoter regions of these key transcription factors during in vitro differentiation of hES cells into thyrocytes. Methods: We initially profiled chromatin accessibility using the technique of Assay for Transposase Accessible Chromatin sequencing (ATAC-seq), and then examined DNA methylation and histone acetylation in the promoter regions of the three selected thyroid transcription factors and the thyroid-specific genes during hES cell differentiation. Results: ATAC-seq analysis showed enriched chromatin accessibility of TAZ, NKX2-1, and PAX8 after exposure to activin A and ethacridine. There were no methylation changes found in the NKX2-1, PAX8, and TAZ promoters by bisulfite sequencing. In contrast, acetylation of histone H4, specifically acetylation of lysine 16, was observed in each of the promoters when measured by chromatin immunoprecipitation polymerase chain reaction assays, which correlated with the activity and expression of NKX2-1 and PAX8 as well as sodium/iodide symporter, thyroid stimulating hormone receptor, and TG genes. Conclusions: These results indicate that ethacridine treatment of activin A-derived endodermal hES cells leads to enhanced chromatin accessibility, which, in turn, allows histone H4 acetylation in the regulation of active genes for speciation of thyroid follicular cells from hES cells.

Understanding Thyroid Cell Stress.

Understanding the regulatory mechanisms that control intracellular stress has fundamental importance since its failure results in cell death. Evidence has emerged indicating that the intracellular signals that are induced in response to diverse stresses include the deoxyribonucleic acid damage response, the unfolded protein response, the mitochondrial and/or endoplasmic reticulum stress responses, and the autophagy signals to degrade dangerous protein aggregates. These signals bring changes to the stressed cells that may support systemic homeostasis or contribute to disease pathology. In normal thyroid cells, both reactive oxygen species (ROS) and antioxidant (AOD) activity is low. An increase in ROS balanced by AOD leads only to mild inflammation, but unopposed increases in ROS lead to a strong inflammatory response and may result in apoptosis. A balance between ROS and AOD is, therefore, needed to maintain thyrocyte homeostasis. This perspective describes how thyroid cells are subjected to multiple insults and how they try to protect themselves using these different cellular responses.

A Stem Cell Surge During Thyroid Regeneration.

Background: Many tissues, including the thyroid, contain resident (adult) stem cells that are responsible for regeneration and repair after injury. The mechanisms of thyroid regeneration and the role of thyroid stem cells and thyroid progenitor cells in this process are not well understood. We have now used a new mouse thyroid injury model to gain insight into this phenomenon. Methods: Tamoxifen induced TPO-Cre mice (TPOCreER2) were crossed with inducible Diphtheria Toxin Receptor homozygous mice (ROSA26iDTR) to give rise to TPOCreER2/iDTR mice, allowing for the Cre-mediated expression of the DTR and rendering TPO expressing thyroid cells highly sensitive to diphtheria toxin (DT). This model of TPOCreER2/iDTR mice allowed us to study the repair/regeneration of thyroid follicles after diphtheria toxin induced thyroid damage by measuring serum thyroid hormones and cell fate. Results: In TPOCreER2/iDTR double transgenic mice we observed severe thyroid damage as early as 2 weeks after initiating intraperitoneal DT injections. There was marked thyroid tissue apoptosis and a ~50% drop in serum T4 levels (from 5.86 to 2.43 ug/dl) and a corresponding increase in serum TSH (from 0.18 to 8.39 ng/dl). In addition, there was a ~50% decrease in transcription of thyroid specific genes (thyroglobulin, TSH receptor, and sodium-iodide symporter). After suspending the DT administration, the thyroid rapidly recovered over a 4-week period during which we observed a transient surge in stem cell marker expression (including Oct4, Nanog, Sox2, and Rex1). In addition, cells immunostaining with stem cell markers Oct4 and Ssea-1 were found in clusters around new thyroid follicles in TPOCreER2/iDTR double transgenic mice. Furthermore, the presence of clusters of thyroid progenitor cells was also identified by Pax8 staining of thyroglobulin negative cells. This recovery of the injured gland was followed by a rapid and sequential restoration of thyroid function. Conclusion: These data demonstrate that a new model of thyroid cell damage induced by DT can be used to study the mobilization of resident adult stem cells. Furthermore, the model clearly demonstrates the involvement of both stem and progenitor cells in the in vivo regeneration of the thyroid after severe destruction.

Cleavage Region Thyrotropin Receptor Antibodies Influence Thyroid Cell Survival In Vivo.

Background: Graves' disease is associated with thyrotropin receptor (TSHR) antibodies of variable bioactivity. Recently, antibodies have been characterized that bind to the cleavage region of the TSHR ectodomain (C-TSHR-Ab), and their ability to induce thyroid cell apoptosis in vitro via excessive cell stress involving multiple organelles was demonstrated. Methods: To investigate the in vivo effects of C-TSHR-Ab, first a murine monoclonal antibody (mAb) directed against residues 337 to 356 of the TSHR cleavage region was developed, and then it was injected into mice. Results: These injections caused reduced serum total triiodothyronine and thyroxine and increased TSH levels compared to control mAb-injected mice. The C-TSHR-mAb induced histological evidence of endoplasmic reticulum stress, mitochondrial stress, and apoptosis in the thyroid glands. C-TSHR-mAb-mediated apoptosis was associated with cellular infiltrates consisting mostly of macrophages, dendritic cells, and neutrophils, while T- and B-lymphocytes were scarce. In addition, in the treated mouse thyroid tissue, hyper-citrullination of histone H3 was also found. This is known to occur via peptidylarginine deiminase 4 and plays an important role in the formation of neutrophil extracellular traps, which are likely to be partly responsible for thyroid infiltration, as seen in many autoimmune diseases. Examination of thyroid tissue from patients with Graves' disease also showed increased stress and some thyrocyte apoptosis compared to normal thyroid tissues. Conclusions: The fact that the C-TSHR-mAb induced accumulation of macrophages, neutrophils, and dendritic cells indicates that innate immunity plays a central role in shaping the adaptive immune response to the TSHR. In addition, this study provides further evidence that the hinge region of the TSHR ectodomain is intimately involved in the immune response in autoimmune thyroid disease.

A Modifying Autoantigen in Graves' Disease.

The TSH receptor (TSHR) is the major autoantigen in Graves' disease (GD). Bioinformatic analyses predict the existence of several human TSHR isoforms from alternative splicing, which can lead to the coexpression of multiple receptor forms. The most abundant of these is TSHRv1.3. In silico modeling of TSHRv1.3 demonstrated the structural integrity of this truncated receptor isoform and its potential binding of TSH. Tissue profiling revealed wide expression of TSHRv1.3, with a predominant presence in thyroid, bone marrow, thymus, and adipose tissue. To gain insight into the role of this v1.3 receptor isoform in thyroid pathophysiology, we cloned the entire open reading frame into a mammalian expression vector. Immunoprecipitation studies demonstrated that both TSHR-stimulating antibody and human TSH could bind v1.3. Furthermore, TSHRv1.3 inhibited the stimulatory effect of TSH and TSHR-Ab MS-1 antibody on TSHR-induced cAMP generation in a dose-dependent manner. To confirm the antigenicity of v1.3, we used a peptide ELISA against two different epitopes. Of 13 GD samples, 11 (84.6%) were positive for a carboxy terminal peptide and 10 (76.9%) were positive with a junction region peptide. To demonstrate that intracellular v1.3 could serve as an autoantigen and modulate disease, we used double-transfected Chinese hamster ovary cells that expressed both green fluorescent protein (GFP)-tagged TSHRv1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR monoclonal antibody and observed the culture supernatant contained v1.3-GFP protein, demonstrating the release of the intracellular receptor variant by this mechanism.