Citations

Introduction There is uncertainty about the number of shark bites on humans in Florida (FL). Methods The International Shark Attack File (ISAF) and Global Shark Attack File (GSAF) databases (2016–2024) were compared with the non-inpatient FL Ambulatory and Emergency Department Patient Data database (AHCA) (2018–2023) and the National Emergency Medical Services (EMS) Information System (NEMSIS) (2016–2024). Results Between 2016–2024 there were 244 FL shark encounters reported in ISAF (mean 27.1 per year), 214 in GSAF (mean 23.8 per year), and 293 in a merged ISAF and GSAF (mean 32.6 per year). Between 2020–2023, there were 373 FL shark encounters reported in AHCA (mean 62.2 per year) compared with 114 encounters reported in NEMSIS (mean 22.8 per year) for 2020–2024. The rate ratio for AHCA FL shark encounters compared to the merged ISAF and GSAF was 1.91 (95% CI 1.64–2.23); p < .001. The percentage of shark encounters in Volusia County, FL, compared with all of FL shark encounters showed no significant differences between the ISAF and GSAF (40.2% and 33.6%, respectively) ( p = .16) and between the AHCA and NEMSIS (7.2% and 7.5%, respectively) ( p = 1.0). However, when comparing either ISAF or GSAF with either AHCA or NEMSIS, there were significant differences ( p < .001 for all). Discussion The AHCA dataset identified about twice as many shark bites as either the ISAF or GSAF. Many FL shark encounters did not require EMS or admission to the hospital. The discrepancy in the number of shark encounters attributed to Volusia County, FL, varied based on the dataset being examined.

Background: Endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of contributing to cardiovascular regeneration. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Hematopoiesis consists of a cascade by which hematopoietic stem cells (HSCs) give rise to either a common myeloid progenitor (CMPs) or common lymphoid progenitor (CLPs). The CMP cells can give rise to either granulocyte-monocyte progenitors (GMPs) or megakaryocyte-erythrocyte progenitors (MEPs). Methods and Results: Herein, we identify that CMPs and GMPs preferentially differentiate into CD34+KDR+ EPCs as measured by FACS, uptake of DiIacLDL and U.lex Lectin, matrigel studies, and endothelial markers. Co-culture studies in matrigel demonstrate increased network formation and incorporation when HUVECs were co-incubated with CMPs and GMPs only. Treatment of CMPs or GMPs with IFN-g blocked EPC differentiation, whereas treatment with TGF-b1 potently induced EPC differentiation. Conversely, EPC formation was markedly impaired using blocking antibodies or TGF-b1 deficient CMPs or GMPs, whereas IFN-g deficient mice have increased EPC differentiation. Functionally, EPCs derived from CMPs and GMPs more potently adhered to fibronectin-coated plates, migrated faster in transwell Boyden chambers, and released VEGF nearly ~2 to 2.5-fold higher than EPCs from MEPs and HSCs. Finally, we identified that a TGF-b1-responsive Kruppel-like transcription factor, KLF10, as robustly expressed ~60-fold in EPCs derived from CMPs and GMPs, compared to hematopoietic progenitors lacking EPC markers. Consistently, KLF10−/− mice have marked defects in EPC differentiation (−23.6% vs. WT, P<0.002), TGF-b1 responsiveness (completedly abolished), and impaired blood flow recovery in response to hindlimb ischemia (−23.8% vs. WT, P<0.008), an effect associated with reduced levels of circulating EPCs (−28.2% vs. WT, P<0.05). Conclusion: Among bone marrow progenitors, a hierarchy exists for EPC differentiation. Targeting KLF10 may alter EPC differentiation from bone marrow-derived CMPs and GMPs and influence cardiovascular repair.

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.

Background— Calcific aortic valve disease is the most common indication for surgical valve replacement in the United States. The cellular mechanisms of valve calcification are not well understood. We have previously shown that cellular proliferation and osteoblastogenesis are important in the development of valvular heart disease. Lrp5, a known low-density receptor-related protein, plays an essential role in cellular proliferation and osteoblastogenesis via the β-catenin signaling pathway. We hypothesize that Lrp5 also plays a role in aortic valve (AV) calcification in experimental hypercholesterolemia. Methods and Results— We examined the effects of cholesterol and atorvastatin in Watanabe rabbits (n=54). Group I (n=18) received a normal diet, group II (n=18) a 0.25% cholesterol diet, and group III (n=18) a 0.25% (w/w) cholesterol diet with atorvastatin for the development of calcification. The AVs were examined for cellular proliferation, Lrp5/β-catenin, and bone matrix markers. Bone formation was assessed by micro-computed tomography, calcein injection, and osteopontin expression. Low-density lipoprotein with and without atorvastatin was also tested in AV myofibroblasts for cellular proliferation and regulation of the Lrp5/β-catenin pathway. Our results demonstrate that the cholesterol diet induced complex bone formations in the calcified AVs with an increase in the Lrp5 receptors, osteopontin, and p42/44 expression. Atorvastatin reduced bone formation, cellular proliferation, and Lrp5/β-catenin protein levels in the AVs. In vitro analysis confirmed the Lrp5/β-catenin expression in myofibroblast cell proliferation. Conclusion— Hypercholesterolemic AV calcification is attenuated by atorvastatin and is mediated in part by the Lrp5/β-catenin pathway. This developmental pathway may be important in the signaling pathway of this disease.

Transforming growth factor (TGF)‐b signaling in naïve T cells induces expression of transcription factor Foxp3, a master regulator in the regulatory T cells (Tregs). However, the molecular mechanisms leading to Foxp3 induction remain unclear. Here we show that Itch−/− T cells are resistant to TGF‐b treatment with reduced Foxp3 expression. The E3 ubiquitin ligase Itch associates with and promotes ubiquitin conjugation to TGF‐b inducible early gene1 product (TIEG1). More importantly, Itch cooperates with TIEG1 to induce Foxp3 expression, which is reversed by TIEG1 deficiency. Functionally TGF‐b‐converted Tregs generated from TIEG1−/− mice are unable to inhibit naïve T cell proliferation in vitro and suppress airway inflammation in vivo. The results suggest an ubiquitin‐dependent non‐proteolytic pathway that is critical for Foxp3 expression and regulation of allergic responses.

Transcriptional control of Runx2 gene expression through two alternative promoters (P1 and P2) is critical for the execution of its function as an osteogenic cell fate determining factor. Using genomic DNaseI footprinting and in vivo DNA binding assays, we identified a helix‐loop‐helix (HLH) motif that overlaps with a Runx motif in the P1 promoter. The HLH mutation decreases P1 transcription 10‐fold in mesenchymal cells, while mutation of the Runx site enhances transcription 2 fold. Although the HLH element is not cell‐type specific, its function may depend on promoter context. Promoter deletion analysis shows that a GA‐rich region (−200 to −128) positively regulates Runx2 P1 transcription and synergizes with the HLH motif. Examination of the −200/−128 region reveals two tandemly repeated GA‐rich elements (GAY motifs) that each contain recognition motifs for purine‐rich DNA binding factors (i.e., SP1 and KLF10/TGFâ Inducible Factor [TIEG]). Forced expression of SP1 or TIEG modulates Runx2 P1 promoter activity through the GAY motifs. Taken together, our data suggest that the Runx2 gene transcription is regulated by synergistic interactions at multiple conserved composite promoter elements to control Runx2 expression and skeletal development.

Abstract A micro version of a nuclear binding assay to assess the biological activity of receptors for steroid hormones was developed for application to small (needle) biopsies of human tumors for the purpose of predicting responses to steroid therapy. This easier assay requires 10-fold less tissue than the original nuclear binding assay described for progesterone receptors in the avian oviduct, endometrium, and endometrial carcinomas (Spelsberg TC, et al., Endocrinology 1987;121:631). We describe the application of this micro assay to normal avian oviduct and cancers of the human breast, and we demonstrate a tissue specificity and saturation of nuclear binding. The micro assay reliably measured as little as 0.5 mg equivalents of tissue per assay tube. Results for breast tumors determined to be estrogen-receptor-positive by the standard dextran-coated charcoal method were also determined with this nuclear binding assay. As described previously for progesterone receptors in endometrial carcinomas, some receptor-positive breast biopsies displayed negligible capacity for nuclear binding. Therefore, with the present assay we have identified nonfunctional receptors in these biopsies, which may be useful for accurate prediction of patients' responses to therapy with hormones.

Total cellular RNA was extracted from bone cells of three different femoral compartments of 2-mo-old rats. The intact femora were first incubated with collagenase to obtain periosteal cells. The bisected periosteum-free diaphyses and metaphyses were then incubated with collagenase to obtain enriched populations of endosteal and cancellous bone cells, respectively. The total cellular RNA from these three tissues was separated by size using agarose gel electrophoresis, transferred to nylon filters, hybridized to 32P-labeled cDNA probes for glyceraldehyde-3-phosphate dehydrogenase (GAP), pre-pro-alpha (I) type I collagen (collagen), osteocalcin (BGP), and alkaline phosphatase (AP), and the cDNA/mRNA hybrids were visualized by radioautography. Bone matrix deposition was measured in each tissue compartment by tetracycline-based dynamic bone histomorphometry. The bone formation and apposition rates were greatest in the periosteum and least in metaphysis. Mean mRNA levels for collagen and BGP were positively correlated with mean bone formation and mineral apposition rates. Interestingly, mean AP mRNA levels were not correlated with indexes of bone formation. These results demonstrate that the steady-state mRNA levels for bone matrix proteins in femora show pronounced site specificity and correlate with the rates of bone matrix deposition.