Chemical Compositions and Experimental and Computational Modeling of the Anticancer Effects of Cnidocyte Venoms of Jellyfish Cassiopea andromeda and Catostylus mosaicus on Human Adenocarcinoma A549 Cells.

Nowadays, major attention is being paid to curing different types of cancers and is focused on natural resources, including oceans and marine environments. Jellyfish are marine animals with the ability to utilize their venom in order to both feed and defend. Prior studies have displayed the anticancer capabilities of various jellyfish. Hence, we examined the anticancer features of the venom of Cassiopea andromeda and Catostylus mosaicus in an in vitro situation against the human pulmonary adenocarcinoma (A549) cancer cell line. The MTT assay demonstrated that both mentioned venoms have anti-tumoral ability in a dose-dependent manner. Western blot analysis proved that both venoms can increase some pro-apoptotic factors and reduce some anti-apoptotic molecules that lead to the inducing of apoptosis in A549 cells. GC/MS analysis demonstrated some compounds with biological effects, including anti-inflammatory, antioxidant and anti-cancer activities. Molecular docking and molecular dynamic showed the best position of each biologically active component on the different death receptors, which are involved in the process of apoptosis in A549 cells. Ultimately, this study has proven that both venoms of C. andromeda and C. mosaicus have the capability to suppress A549 cells in an in vitro condition and they might be utilized in order to design and develop brand new anticancer agents in the near future.

Tissue Extract from Brittle Star Undergoing Arm Regeneration Promotes Wound Healing in Rat.

This study set out to evaluate the wound healing properties of brittle star extracts in vitro and in vivo. Due to the great arm regeneration potential of the brittle star, Ophiocoma cynthiae, the present study aimed to evaluate the wound healing effect of hydroalcoholic extracts of brittle star undergoing arm regeneration in wound healing models. The brittle star samples were collected from Nayband Bay, Bushehr, Iran. After wound induction in the arm of brittle stars, hydroalcoholic extracts relating to different times of arm regeneration were prepared. The GC-MS analysis, in vitro MTT cell viability and cell migration, Western blot, and computational analysis tests were performed. Based on the in vitro findings, two BSEs were chosen for in vivo testing. Macroscopic, histopathological and biochemical evaluations were performed after treatments. The results showed positive proliferative effects of BSEs. Specifically, forty-two compounds were detected in all groups of BSEs using GC-MS analysis, and their biological activities were assessed. The MTT assay showed that the 14 d BSE had a higher proliferative effect on HFF cells than 7 d BSE. The cell migration assay showed that the wound area in 7 d and 14 d BSEs was significantly lower than in the control group. Western blot analysis demonstrated an increase in the expression of proliferation-related proteins. Upon the computational analysis, a strong affinity of some compounds with proteins was observed. The in vivo analysis showed that the evaluation of wound changes and the percentage of wound healing in cell migration assay in the 7 d BSE group was better than in the other groups. Histopathological scores of the 7 d BSE and 14 d BSE groups were significantly higher than in the other groups. In conclusion, the hydroalcoholic extract of O. cynthiae undergoing arm regeneration after 7 and 14 days promoted the wound healing process in the cell and rat skin wound healing model due to their proliferative and migratory biological activity.

Proliferative Effect of Aqueous Extract of Sea Cucumber (Holothuria parva) Body Wall on Human Umbilical Cord Mesenchymal Stromal/Stem Cells.

Sea cucumber extracts and their bioactive compounds have the potential for stem cell proliferation induction and for their beneficial therapeutic properties. In this study, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were exposed to an aqueous extract of Holothuria parva body walls. Proliferative molecules were detected using gas chromatography-mass spectrometry (GC-MS) analysis in an aqueous extract of H. parva. The aqueous extract concentrations of 5, 10, 20, 40, and 80 µg/mL and 10 and 20 ng/mL of human epidermal growth factor (EGF) as positive controls were treated on hUC-MSCs. MTT, cell count, viability, and cell cycle assays were performed. Using Western blot analysis, the effects of extracts of H. parva and EGF on cell proliferation markers were detected. Computational modeling was done to detect effective proliferative compounds in the aqueous extract of H. parva. A MTT assay showed that the 10, 20, and 40 µg/mL aqueous extract of H. parva had a proliferative effect on hUC-MSCs. The cell count, which was treated with a 20 µg/mL concentration, increased faster and higher than the control group (p < 0.05). This concentration of the extract did not have a significant effect on hUC-MSCs' viability. The cell cycle assay of hUC-MSCs showed that the percentage of cells in the G2 stage of the extract was biologically higher than the control group. Expression of cyclin D1, cyclin D3, cyclin E, HIF-1α, and TERT was increased compared with the control group. Moreover, expression of p21 and PCNA decreased after treating hUC-MSCs with the extract. However, CDC-2/cdk-1 and ERK1/2 had almost the same expression as the control group. The expression of CDK-4 and CDK-6 decreased after treatment. Between the detected compounds, 1-methyl-4-(1-methyl phenyl)-benzene showed better affinity to CDK-4 and p21 than tetradecanoic acid. The H. parva aqueous extract showed proliferative potential on hUC-MSCs.

Decellularization of kidney tissue: comparison of sodium lauryl ether sulfate and sodium dodecyl sulfate for allotransplantation in rat.

An automatic decellularization device was developed to perfuse and decellularize male rats' kidneys using both sodium lauryl ether sulfate (SLES) and sodium dodecyl sulfate (SDS) and to compare their efficacy in kidney decellularization and post-transplantation angiogenesis. Kidneys were perfused with either 1% SDS solution for 4 h or 1% SLES solution for 6 h. The decellularized scaffolds were stained with hematoxylin and eosin, periodic acid Schiff, Masson's trichrome, and Alcian blue to determine cell removal and glycogen, collagen, and glycosaminoglycan contents, respectively. Moreover, scanning electron microscopy was performed to evaluate the cell removal and preservation of microarchitecture of both SDS and SLES scaffolds. Additionally, DNA quantification assay was applied for all groups in order to measure residual DNA in the scaffolds and normal kidney. In order to demonstrate biocompatibility of the decellularized scaffolds, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were seeded on the scaffolds. In addition, the allotransplantation was performed in back muscle and angiogenesis was evaluated. Complete cell removal in both SLES and SDS groups was observed in scanning electron microscopy and DNA quantification assays. Moreover, the extracellular matrix (ECM) architecture of rat kidney in the SLES group was significantly preserved better than the SDS group. The hUC-MSCs were successfully migrated from the cell culture plate surface into the SDS and SLES decellularized scaffolds. The formation of blood vessels was observed in the kidney in both SLES and SDS decellularized kidneys. The better preservation of ECM than SDS introduces SLES as the solvent of choice for kidney decellularization.

Socialization During the COVID-19 Pandemic: The Role of Social and Scientific Networks During Social Distancing.

In the COVID-19 era, while we are encouraged to be physically far away from each other, social and scientific networking is needed more than ever. The dire consequences of social distancing can be diminished by social networking. Social media, a quintessential component of social networking, facilitates the dissemination of reliable information and fighting against misinformation by health authorities. Distance learning, telemedicine, and telehealth are among the most prominent applications of networking during this pandemic. Additionally, the COVID-19 pandemic highlights the importance of collaborative scientific efforts. In this chapter, we summarize the advantages of harnessing both social and scientific networking in minimizing the harms of this pandemic. We also discuss the extra collaborative measures we can take in our fight against COVID-19, particularly in the scientific field.

Teaching mid-urethral sling surgery to residents: Impact on operative time and postoperative outcomes.

AIMS: The purpose of this study was to determine the impact of resident teaching on outcomes of mid-urethral sling surgery. METHODS: A retrospective review of female patients who underwent an outpatient transobturator (TOT) synthetic mid-urethral sling procedure with and without concomitant prolapse repair by two surgeons (JA, KE) in a tertiary female pelvic medicine practice was performed. Total procedure time (TPT = time from incision to closure including sling placement and any prolapse procedure), estimated blood loss (EBL), and postoperative complications including urinary retention, mesh exposure, reoperation, vaginal bleeding, and leg pain were compared between cases with and without the presence of a resident. RESULTS: One hundred thirty-four women underwent an outpatient transobturator sling procedure. Fifty-seven patients (43%) had a concomitant prolapse procedure. A resident was present at 57% (76/134) of cases. The average observed TPT (±SEM) was 60.6 ± 3.1 min when a resident was present and 46.6 ± 2.5 min when a resident was not present (P = 0.001). However, residents were more likely to be present when concomitant procedures were performed (P = 0.003). After adjusting for this, the presence of a resident increased TPT by an estimated 7.9 ± 2.5 min (P = 0.002). There was no statistical difference in EBL or postoperative complications. CONCLUSIONS: Resident participation in transobturator sling procedures resulted in a statistically significant, although clinically small, increase in operative time and had no significant impact on EBL or postoperative complications.

3D high-resolution diffusion-weighted MRI at 3T: Preliminary application in prostate cancer patients undergoing active surveillance protocol for low-risk prostate cancer.

PURPOSE: To improve spatial resolution and image quality of diffusion-weighted (DW) MRI in detecting low-risk prostate cancer (lrPC) in patients undergoing active surveillance protocol (AS-PC), we propose the application of a diffusion-prepared balanced steady-state free precession (bSSFP) technique capable of multishot acquisition. METHODS: Diffusion-prepared bSSFP was compared with single-shot DW echo planar imaging (SS-DW-EPI) at two prescribed resolutions (2.1 × 2.1 × 3.5mm(3) , 0.9 × 0.9 × 3.5 mm(3) ) in nine healthy subjects and nine AS-PC patients. Geometric distortion and susceptibility artifacts were quantitatively assessed in all subjects. In AS-PC patients, lesion detection via blinded multiparametric MRI including T1-weighted, T2-weighted, dynamic contrast-enhanced imaging, and along with either of two DW methods were evaluated against 12-point biopsy. RESULTS: Geometric distortion and susceptibility artifacts were significantly less for diffusion-prepared bSSFP at both prescribed spatial resolutions than SS-DW-EPI. Apparent diffusion coefficients of healthy prostate tissue were concordant between the two DW methods at both spatial resolutions. In AS-PC patients, multiparametric MRI with diffusion-prepared bSSFP had greater sensitivity (94%, 63%), accuracy (76%, 67%), positive-predictive value (54%, 48%), negative-predictive value (97%, 82%), and area under the curve (0.80, 0.67) than with SS-DW-EPI. CONCLUSIONS: The proposed diffusion-prepared technique with higher spatial resolution and improved image quality over SS-DW-EPI resulted in better multiparametric MRI detection of lrPC in AS-PC patients.

Impact of post prostate biopsy hemorrhage on multiparametric magnetic resonance imaging.

INTRODUCTION: Hemorrhage induced by prostate biopsy can interfere with the interpretation of prostate magnetic resonance imaging (MRI). MATERIALS AND METHODS: We reviewed 101 patients who had prostate multiparametric MRI (MP-MRI) and radical prostatectomy. RESULTS: On MRI obtained within 4 weeks following the biopsy, hemorrhage was seen in 26/36 (72.2%) patients. Patients having a MRI between 4-6 weeks of the biopsy had hemorrhage in 8/14 (57.1%) cases. After 6 weeks, hemorrhage was less common but still present in 24/46 (52%) patients. There were five patients who had prostate MRI prior to biopsy and served as a control group. There was no significant correlation between the length of time beyond 6 weeks and the likelihood of having prostate hemorrhage on MRI. The overall sensitivity and specificity of MRI for predicting extracapsular extension (ECE) were 78.6% and 89%, respectively. However, if the analysis was limited to patients with MRI within 6 weeks from the time of biopsy, the sensitivity and specificity were similar: 80% and 90%, respectively. For patients with MRI obtained after 6 weeks, the sensitivity and specificity were 76.9% and 87.9%. CONCLUSIONS: Prostate hemorrhage is seen in the majority of cases within 6 weeks of biopsy and can be seen in nearly half the patients even beyond 6 weeks. However, hemorrhage within 6 weeks of a biopsy does not interfere with assessment for ECE.

Improving performance, reproduction, and immunity in laying Japanese quail with algal derivatives.

We investigated the effect of the Persian Gulf algae derivatives, namely phycocyanin (PC) and fucoidan (FUC), on the performance, reproductive traits, and immune responses of laying Japanese quails. A completely randomized design was used to distribute 250 six-wk-old Japanese quails with an average body weight of 215 ± 10 g into 5 treatments, 5 replicates, and 10 birds in each replicate over a 5-wk period. Unlike the control groups, the treatment groups received drinking water supplemented with PC and FUC at concentrations of 20 or 40 mg/L, denoted as PC20, PC40, FUC20, and FUC40, respectively, while all birds were provided with identical feed. Supplemental algal derivatives notably improved hen day egg production (HDEP), egg mass, and feed conversion ratio (FCR) compared to the control group (P < 0.01). Incorporating PC and FUC had no significant effect on the weight of males' testes or the weight and length of hens' oviducts. Additionally, the experimental treatments had no impact on the chicks' hatching weight. The supplementation of PC and FUC resulted in increased fertility (P = 0.038) and hatchability (P < 0.001) rates, with the exception of fertility in the PC40 group. The effect of the experimental treatments on immune responses was largely not statistically significant, except in the case of ND. Specifically, the experimental treatments resulted in increased (P = 0.033) antibody titers against ND when compared to the control group, with the exception of FUC20. Supplemental algal derivatives significantly (P < 0.01) reduced total cholesterol, creatinine, and triglycerides (except in the case of PC20). Overall, these findings underscore the potential of algal derivatives to enhance quail performance, reproductive traits, and immune responses.

Tissue clearing and three-dimensional imaging of intact tissues: a review on FACT protocol.

FACT is a developed technique for clearing tissues that does not use acrylamide. Since the removal of lipids is crucial for transparency and efficient antibody staining throughout the tissue, especially for microscopy and imaging, it is a harmful process that can cause the loss of important biological molecules such as proteins. The FACT technique overcomes this by chemically bonding the membrane and intracellular proteins with the extracellular matrix, creating a massive 3D hydrogel matrix and providing structural support to fortify the tissue during processing. Compared to other acrylamide-based techniques, the FACT technique requires less labor and harmful chemicals and is therefore considered a more suitable option. In this study, we describe the complete FACT protocol for antibody staining and imaging of whole-cleared tissues while preserving structure and improving image quality. The protocol includes tissue perfusion, fixation, clearing, antibody staining, refractive index matching (RIM) (), microscopy, and imaging. The timing for each step varies depending on the size, weight, and type of tissue, as well as the type of immunostaining. We provide an example of the FACT protocol using mouse brain tissue, which demonstrates its suitability for molecular interrogation analysis of large tissues. The FACT technique has been successfully performed on different types of tissues, making it a favorable choice for a variety of applications.

Proliferating and migrating effects of regenerating sea anemone Aulactinia stella cells-derived exosomes on human skin fibroblasts.

Regenerative effects of sea anemone-derived exosomes on human foreskin fibroblasts (HFFs) were investigated. Water-based extracts from regenerating Aulactinia stella tissue were collected at various time points, and exosomes were extracted after inducing wounds. Both the extract and exosomes were tested on HFF for proliferation and in vitro wound healing. In silico analysis explored protein-protein docking between regenerative exosome proteins and HFF receptors. The MTT (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide proliferation assay and in vitro wound healing test of aquatic extract showed proliferative effects on HFF cell lines, with the 60 µg/mL concentration significantly enhancing cell migration. Exosomes were characterised. Exosomes showed a significantly positive effect on cell proliferation and migration at the 50 µg/mL concentration 48 h post-wound induction. In silico analysis revealed potential binding affinities between exosome proteins and HFF receptors. In conclusion, optimised concentrations of A. stella-derived exosomes exhibited positive effects on HFF regeneration and migration, suggesting their potential in accelerating wound healing.

Effects of Different Coatings, Primers, and Additives on Corrosion of Steel Rebars.

In this research, methods of increasing the corrosion resistance of reinforced concrete were experimentally investigated. The study used silica fume and fly ash at optimized percentages of 10 and 25% by cement weight, polypropylene fibers at a ratio of 2.5% by volume of concrete, and a commercial corrosion inhibitor, 2-dimethylaminoethanol (Ferrogard 901), at 3% by cement weight. The corrosion resistance of three types of reinforcements, mild steel (STt37), AISI 304 stainless steel, and AISI 316 stainless steel, was investigated. The effects of various coatings, including hot-dip galvanizing, alkyd-based primer, zinc-rich epoxy primer, alkyd top coating, polyamide epoxy top coating, polyamide epoxy primer, polyurethane coatings, a double layer of alkyd primer and alkyd top coating, and a double layer of epoxy primer and alkyd top coating, were evaluated on the reinforcement surface. The corrosion rate of the reinforced concrete was determined through results of accelerated corrosion and pullout tests of steel-concrete bond joints and stereographic microscope images. The samples containing pozzolanic materials, the corrosion inhibitor, and a combination of the two showed significant improvement in corrosion resistance by 7.0, 11.4, and 11.9 times, respectively, compared to the control samples. The corrosion rate of mild steel, AISI 304, and AISI 316 decreased by 1.4, 2.4, and 2.9 times, respectively, compared to the control sample; however, the presence of polypropylene fibers reduced the corrosion resistance by 2.4 times compared to the control.

MRI Tracking of Marine Proliferating Cells In Vivo Using Anti-Oct4 Antibody-Conjugated Iron Nanoparticles for Precision in Regenerative Medicine.

Marine invertebrates are multicellular organisms consisting of a wide range of marine environmental species. Unlike vertebrates, including humans, one of the challenges in identifying and tracking invertebrate stem cells is the lack of a specific marker. Labeling stem cells with magnetic particles provides a non-invasive, in vivo tracking method using MRI. This study suggests antibody-conjugated iron nanoparticles (NPs), which are detectable with MRI for in vivo tracking, to detect stem cell proliferation using the Oct4 receptor as a marker of stem cells. In the first phase, iron NPs were fabricated, and their successful synthesis was confirmed using FTIR spectroscopy. Next, the Alexa Fluor anti-Oct4 antibody was conjugated with as-synthesized NPs. Their affinity to the cell surface marker in fresh and saltwater conditions was confirmed using two types of cells, murine mesenchymal stromal/stem cell culture and sea anemone stem cells. For this purpose, 106 cells of each type were exposed to NP-conjugated antibodies and their affinity to antibodies was confirmed by an epi-fluorescent microscope. The presence of iron-NPs imaged with the light microscope was confirmed by iron staining using Prussian blue stain. Next, anti-Oct4 antibodies conjugated with iron NPs were injected into a brittle star, and proliferating cells were tracked by MRI. To sum up, anti-Oct4 antibodies conjugated with iron NPs not only have the potential for identifying proliferating stem cells in different cell culture conditions of sea anemone and mouse cell cultures but also has the potential to be used for in vivo MRI tracking of marine proliferating cells.

Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold.

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Algal Cells-Derived Extracellular Vesicles: A Review With Special Emphasis on Their Antimicrobial Effects.

Extracellular vesicles (EVs) originated from different cells of approximately all kinds of organisms, recently got more attention because of their potential in the treatment of diseases and reconstructive medicine. To date, lots of studies have been performed on mammalian-derived vesicles, but little attention has been paid to algae and marine cells as valuable sources of EVs. Proving the promising role of EVs in medicine requires sufficient resources to produce qualified microvesicles. Algae, same as its other sister groups, such as plants, have stem cells and stem cell niches. Previous studies showed the EVs in plants and marine cells. So, this study was set out to talk about algal extracellular vesicles. EVs play a major role in cell-to-cell communication to convey molecules, such as RNA/DNA, metabolites, proteins, and lipids within. The components of EVs depends on the origin of the primitive cells or tissues and the isolation method. Sufficient resources are needed to produce high-quality, stable, and compatible EVs as a drug or drug delivery system. Plant stem cells have great potential as a new controllable resource for the production of EVs. The EVs secreted from stem cells can easily be extracted from the cell culture medium and evaluated for medicinal uses. In this review, the aim is to introduce algae stem cells as well as EVs derived from algal cells. In the following, the production of the EVs¸ the properties of EVs extracted from these sources and their antimicrobial effects will be discussed.

Assessment of Quality of Life and the Risk of Obstructive Sleep Apnea in Individuals with Chronic Rhinosinusitis who are Candidate for Functional Endoscopic Sinus Surgery.

BACKGROUND: Chronic rhinosinusitis is associated with changes in quality of life (QoL). The present study intended to evaluate the QoL and risk of obstructive sleep apnea in individuals with chronic rhinosinusitis who were candidate for functional endoscopic sinus surgery. To determine the Quality of Life and the risk of sleep apnea in cases with chronic Rhinosinusitis. MATERIALS AND METHODS: A total of 100 patients with drug-resistant chronic rhinosinusitis candidate for functional endoscopic sinus surgery referred to the ENT clinic of Masih Daneshvari Hospital, Tehran, Iran were recruited. SNOT-22 and STOP-BANG questionnaires were filled before the surgery. RESULTS: The mean SNOT-22 score was 40.44, with a standard deviation of 19.27 (ranged from 1 to 94). Also, according to the STOP-BANG questionnaire, 62% of participants were at increased risk of OSA. Based on the cut-off point of 30 for the SNOT-22 score (either larger or lower than 30), patients were categorized into two groups. Sixty-eight percent of participants were categorized in ≥ 30 SNOT-22 score. Age below 50, female gender, and those at high risk of OSA were associated with lower QoL. CONCLUSION: Most patients with chronic rhinosinusitis had a low QoL and were mostly at increased risk of OSA. Being women younger than 50 years and the presence of OSA probably are associated with lower QoL in these patients.

Three-dimensional and two-dimensional relationships of gangliogenesis with folliculogenesis in mature mouse ovary: a Golgi-Cox staining approach.

The present study was set out to investigate two-dimensional (2D) and three-dimensional (3D) evaluations of ovarian nervous network development and the structural relationship between folliculogenesis and gangliogenesis in mouse ovaries. Adult mice ovarian tissue samples were collected from follicular and luteal phases after cardiac perfusion. Ovarian samples were stained by a Golgi-Cox protocol. Following staining, tissues were serially sectioned for imaging. Neural filaments and ganglia were present in the ovaries. In both 2D and 3D studies, an increase in the number and area of ganglia was seen during the follicular growth. The same pattern was also seen in corpora lutea development. However, in some cases such as ratio of ganglia number to follicle area, the ratio of ganglia area to follicular area, 2D findings were different compared with the 3D results. 3D analysis of ovarian gangliogenesis showed the possible direct effect of them on folliculogenesis. Golgi-Cox staining was used in this study for 3D evaluation in non-brain tissue. The results of 3D analysis of the present study showed that, in some cases, the information provided by 2D analysis does not match the reality of ovarian neuronal function. This confirmed the importance of 3D analysis for evaluation of ovarian function.

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF-beta signaling.

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-beta signaling directed the prostatic induction. UGM from TGF-beta type II receptor stromal conditional knockout mice (Tgfbr2(fspKO)) or control mice (Tgfbr2(floxE2/floxE2)) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2(floxE2/floxE2) UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2(fspKO) UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2(fspKO) UGM compared with Tgfbr2(floxE2/floxE2) UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-beta activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-beta signaling to mediate epithelial Wnt activity.

Temporal-spatial protein expression in bladder tissue derived from embryonic stem cells.

PURPOSE: Identifying developmental proteins could lead to markers of bladder progenitor cells, which could be used to investigate bladder diseases. We recently reported a novel embryonic stem cell model in which to study differential protein expression patterns during bladder development. Differential and temporal expressions of the endodermal proteins known as forkhead box (Foxa1 and Foxa2) were observed. In the current study we further delineated these protein expression patterns. MATERIALS AND METHODS: Epithelium was removed from the underlying mesenchyma from embryonic day 18 rat bladders. Heterospecific recombinant xenografts were created by combining embryonic stem cells plus embryonic bladder mesenchyma and placed beneath the renal capsule of mouse hosts. Grafts were harvested at 16, 18, 21, 28, 35 and 42 days, and evaluated with hematoxylin and eosin, trichrome staining, and immunohistochemistry for uroplakin, smooth muscle alpha-actin, p63, Foxa1, Foxa2 and androgen receptor. RESULTS: At 16 days uroplakin was detectable and it seemed to correlate with the loss of Foxa2, while Foxa1 remained at all time points. Androgen receptor was first noted in stroma at day 16. It localized to urothelial nuclei at day 21 and was undetectable at 42 days. Adjacent to the urothelium alpha-smooth muscle actin was seen on day 16 and it was localized in bundles to the periphery of the graft at later time points. Staining for basilar urothelium with p63 confirmed basilar orientation at all time points. CONCLUSIONS: We report the temporal spatial expression of various genes in early bladder development. This suggests that some proteins may be potential markers of bladder progenitor cells. Characterizing these markers may potentially identify bladder progenitor cells that have been directed toward a lineage path destined to become urothelial cells. Ultimately these multipotential progenitor cells could be isolated and used to study and treat diseases that affect the bladder.

Effect of conditional knockout of the type II TGF-beta receptor gene in mammary epithelia on mammary gland development and polyomavirus middle T antigen induced tumor formation and metastasis.

Transforming growth factor-beta (TGF-beta) isoforms are growth factors that function physiologically to regulate development, cellular proliferation, and immune responses. The role of TGF-beta signaling in mammary tumorigenesis is complex, as TGF-beta has been reported to function as both a tumor suppressor and tumor promoter. To elucidate the role of TGF-beta signaling in mammary gland development, tumorigenesis, and metastasis, the gene encoding type II TGF-beta receptor, Tgfbr2, was conditionally deleted in the mammary epithelium (Tgfbr2MGKO). Loss of Tgfbr2 in the mammary epithelium results in lobular-alveolar hyperplasia in the developing mammary gland and increased apoptosis. Tgfbr2MGKO mice were mated to the mouse mammary tumor virus-polyomavirus middle T antigen (PyVmT) transgenic mouse model of metastatic breast cancer. Loss of Tgfbr2 in the context of PyVmT expression results in a shortened median tumor latency and an increased formation of pulmonary metastases. Thus, our studies support a tumor-suppressive role for epithelial TGF-beta signaling in mammary gland tumorigenesis and show that pulmonary metastases can occur and are even enhanced in the absence of TGF-beta signaling in the carcinoma cells.