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A strategy for cancer treatment was implemented, based on chemo-photodynamic therapy, utilizing a novel formulation, low-cost system called Cas-ZnONPs. This system consisted of the incorporation of Casiopeina III-ia (CasIII-ia), a hydrophilic copper coordination compound with well-documented anti-neoplastic activity, on Zinc oxide nanoparticles (ZnONPs) with apoptotic activity and lipophilicity, allowing them to permeate biological barriers. Additionally, ZnONPs exhibited fluorescence, with emission at different wavelengths depending on their agglomeration and enabling real-time tracking biodistribution. Also, ZnONPs served as a sensitizer, generating reactive oxygen species (ROS) in situ. In in vitro studies on HeLa and MDA-MB-231 cell lines, a synergistic effect was observed with the impregnated CasIII-ia on ZnONPs. The anticancer activity had an increase in cellular inhibition, depending on the dose of exposure to UV-vis irradiation. In in vivo studies utilized zebrafish models for xenotransplanting stained MDA-MB-231 cells and testing the effectiveness of Cas-ZnONPs treatment. The treatment successfully eliminated cancer cells, both when combined with Photodynamic Therapy (PDT) and when used alone. However, a significantly higher concentration (50 times) of Cas-ZnONPs was required in the absence of PDT. This demonstrates the potential of Cas-ZnONPs in cancer treatment, especially when combined with PDT., Competing Interests: Declaration of competing interest All Authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastric cancer is the fourth leading cause of cancer deaths worldwide. The presence of chemoresistant cells has been used to explain this high mortality rate. These higher tumorigenic and chemoresistant cells involve cancer stem cells (CSCs), which have the potential for self-renewal, a cell differentiation capacity, and a greater tumorigenic capacity. Our research group identified gastric cancer stem cells (GCSCs) with the CD24+CD44+CD326+ICAM1+ immunophenotype isolated from gastric cancer patients. Interestingly, this GCSC immunophenotype was absent in cells isolated from healthy people, who presented a cell population with a CD24+CD44+CD326+ immunophenotype, lacking ICAM1. We aimed to explore the role of ICAM1 in these GCSCs; for this purpose, we isolated GCSCs from the AGS cell line and generated a GCSC line knockout for ICAM1 using CRISPR/iCas9, which we named GCSC-ICAM1 KO . To assess the role of ICAM1 in the GCSCs, we analyzed the migration, invasion, and chemoresistance capabilities of the GCSCs using in vitro assays and evaluated the migratory, invasive, and tumorigenic properties in a zebrafish model. The in vitro analysis showed that ICAM1 regulated STAT3 activation (pSTAT3-ser727) in the GCSCs, which could contribute to the ability of GCSCs to migrate, invade, and metastasize. Interestingly, we demonstrated that the GCSC-ICAM1 KO cells lost their capacity to migrate, invade, and metastasize, but they exhibited an increased resistance to a cisplatin treatment compared to their parental GCSCs; the GCSC-ICAM1 KO cells also exhibited an increased tumorigenic capability in vivo.

Betaglycan (BG) is a transmembrane co-receptor of the transforming growth factor-β (TGF-β) family of signaling ligands. It is essential for embryonic development and tissue homeostasis and fertility in adults. It functions by enabling binding of the three TGF-β isoforms to their signaling receptors and is additionally required for inhibin A (InhA) activity. Despite its requirement for the functions of TGF-βs and InhA in vivo, structural information explaining BG ligand selectivity and its mechanism of action is lacking. Here, we determine the structure of TGF-β bound both to BG and the signaling receptors, TGFBR1 and TGFBR2. We identify key regions responsible for ligand engagement, which has revealed novel binding interfaces that differ from those described for the closely related co-receptor of the TGF-β family, endoglin, thus demonstrating remarkable evolutionary adaptation to enable ligand selectivity. Finally, we provide a structural explanation for the hand-off mechanism underlying TGF-β signal potentiation.

Persistent HGF/Met signaling drives tumor growth and dissemination. Proteoglycans within the tumor microenvironment might control HGF availability and signaling by affecting its accessibility to Met (HGF receptor), likely defining whether acute or sustained HGF/Met signaling cues take place. Given that betaglycan (BG, also known as type III TGFβ receptor or TGFBR3), a multi-faceted proteoglycan TGFβ co-receptor, can be found within the tumor microenvironment, we addressed its hypothetical role in oncogenic HGF signaling. We found that HGF/Met promotes lung cancer and endothelial cells migration via PI3K and mTOR. This effect was enhanced by recombinant soluble betaglycan (solBG) via a mechanism attributable to its glycosaminoglycan chains, as a mutant without them did not modulate HGF effects. Moreover, soluble betaglycan extended the effect of HGF-induced phosphorylation of Met, Akt, and Erk, and membrane recruitment of the RhoGEF P-Rex1. Data-mining analysis of lung cancer patient datasets revealed a significant correlation between high MET receptor, HGF, and PREX1 expression and reduced patient survival. Soluble betaglycan showed biochemical interaction with HGF and, together, they increased tumor growth in immunocompetent mice. In conclusion, the oncogenic properties of the HGF/Met pathway are enhanced and sustained by GAG-containing soluble betaglycan., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Mycobacterium tuberculosis is the main causal agent of pulmonary tuberculosis (TB); the treatment of this disease is long and involves a mix of at least four different antibiotics that frequently lead to abandonment, favoring the surge of drug-resistant mycobacteria (MDR-TB), whose treatment becomes more aggressive, being longer and more toxic. Thus, the search for novel strategies for treatment that improves time or efficiency is of relevance. In this work, we used a murine model of pulmonary TB produced by the MDR-TB strain to test the efficiency of gene therapy with adenoviral vectors codifying TNF (AdTNF), a pro-inflammatory cytokine that has protective functions in TB by inducing apoptosis, granuloma formation and expression of other Th1-like cytokines. When compared to the control group that received an adenoviral vector that codifies for the green fluorescent protein (AdGFP), a single dose of AdTNF at the chronic active stage of the disease produced total survival, decreasing bacterial load and tissue damage (pneumonia), which correlated with an increase in cells expressing IFN-γ, iNOS and TNF in pneumonic areas and larger granulomas that efficiently contain and eliminate mycobacteria. Second-line antibiotic treatment against MDR-TB plus AdTNF gene therapy reduced bacterial load faster within a week of treatment compared to empty vector plus antibiotics or antibiotics alone, suggesting that AdTNF is a new potential type of treatment against MDR-TB that can shorten second-line chemotherapy but which requires further experimentation in other animal models (non-human primates) that develop a more similar disease to human pulmonary TB.

Background: Betaglycan, also known as the TGFβ type III receptor (Tgfbr3), is a co-receptor that modulates TGFβ family signaling. Tgfbr3 is upregulated during C2C12 myoblast differentiation and expressed in mouse embryos myocytes., Results: To investigate tgfbr3 transcriptional regulation during zebrafish embryonic myogenesis, we cloned a 3.2 kb promoter fragment that drives reporter transcription during C2C12 myoblasts differentiation and in the Tg(tgfbr3:mCherry) transgenic zebrafish. We detect tgfbr3 protein and mCherry expression in the adaxial cells concomitantly with the onset of their radial migration to become slow-twitch muscle fibers in the Tg(tgfbr3:mCherry). Remarkably, this expression displays a measurable antero-posterior somitic gradient expression., Conclusions: tgfbr3 is transcriptionally regulated during somitic muscle development in zebrafish with an antero-posterior gradient expression that preferentially marks the adaxial cells and their descendants., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Lipid droplets (LDs) are intracellular organelles found in most cell types from adipocytes to cancer cells. Although recent investigations have implicated LDs in numerous diseases, the current available methods to monitor them in vertebrate models rely on static imaging using fluorescent dyes, limiting the investigation of their rapid in vivo dynamics. Here, we report a fluorophore chemistry approach to enable in vivo LD dynamic monitoring using a Nernstian partitioning mechanism. Interestingly, the effect of atorvastatin and osmotic treatments toward LDs revealed an unprecedented dynamic enhancement. Then, using a designed molecular probe with an optimized response to hydration and LD dynamics applied to Zebrafish developing pericardial and yolk-sac edema, which represents a tractable model of a human cardiovascular disease, we also provide a unique dual method to detect disease evolution and recovery.

Genistein is an isoflavone present in soybeans and is considered a bioactive compound due to its widely reported biological activity. We have previously shown that intraperitoneal genistein administration and diet supplementation activates the thermogenic program in rats and mice subcutaneous white adipose tissue (scWAT) under multiple environmental cues, including cold exposure and high-fat diet feeding. However, the mechanistic insights of this process were not previously unveiled. Uncoupling protein 1 (UCP1), a mitochondrial membrane polypeptide responsible for dissipating energy into heat, is considered the most relevant thermogenic marker; thus, we aimed to evaluate whether genistein regulates UCP1 transcription. Here we show that genistein administration to thermoneutral-housed mice leads to the appearance of beige adipocyte markers, including a sharp upregulation of UCP1 expression and protein abundance in scWAT. Reporter assays showed an increase in UCP1 promoter activity after genistein stimulation, and in silico analysis revealed the presence of estrogen (ERE) and cAMP (CRE) response elements as putative candidates of genistein activation. Mutation of the CRE but not the ERE reduced genistein-induced promoter activity by 51%. Additionally, in vitro and in vivo ChIP assays demonstrated the binding of CREB to the UCP1 promoter after acute genistein administration. Taken together, these data elucidate the mechanism of genistein-mediated UCP1 induction and confirm its potential applications in managing metabolic disorders., (© 2023 Federation of American Societies for Experimental Biology.)

Background: Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Specific and thorough identification of cancer cell subsets with higher tumorigenicity and chemoresistance, such as cancer stem cells (CSCs), could lead to the development of new and promising therapeutic targets. For better CSC identification, a complete or extended surface marker phenotype is needed to provide increased specificity for new cell targeting approaches. Our goal is to identify and characterize a putative extended phenotype for CSCs derived from patients with GC before treatment, as well as to evaluate its clinical value. In addition, we aim to ensure that cells with this phenotype have stemness and self-renewal capabilities., Methods: This is a cohort study including 127 treatment-naïve patients with GC who attended the Instituto Nacional de Cancerología. Multiparametric flow cytometry analysis was performed to determine the extended phenotype of cells derived from gastric biopsies. The tumorigenic capability of cells identified in patients was assessed in a zebrafish model., Results: CD24+CD44+CD54+EpCAM+ cells were present in all treatment-naïve patients included, with a median abundance of 1.16% (0.57-1.89%). The percentage of CD24+CD44+CD54+EpCAM+ cells was categorized as high or low using 1.19% as the cutoff for the CD24+CD44+CD54+EpCAM+ cell subset. Additionally, a higher TNM stage correlated with a higher percentage of CD24+CD44+CD54+EpCAM+ cells (Rho coefficient 0.369; p < 0.0001). We also demonstrated that a higher percentage of CD24+CD44+CD54+EpCAM+ cells was positively associated with metastasis. The metastatic potential of these cells was confirmed in a zebrafish model. Ultimately, under our conditions, we conclude that CD24+CD44+CD54+EpCAM+ cells are true gastric cancer stem cells (GCSCs)., Conclusion: The CD24+CD44+CD54+EpCAM+ cells present in tissue samples from patients are true GCSCs. This extended phenotype results in better and more specific characterization of these highly tumorigenic cells. The relative quantification of CD24+CD44+CD54+EpCAM+ cells has potential clinical value, as these cells are associated with metastatic disease, making their presence an additional prognostic marker and possibly a target for the design of new antineoplastic treatments in the era of precision oncology. Overall, the extended CD24+CD44+CD54+EpCAM+ phenotype of GCSCs could support their isolation for the study of their stemness mechanisms, leading to the identification of better molecular targets for the development of both new therapeutic approaches such as oncoimmunotherapy and new diagnostic and clinical prognostic strategies for GC., (© 2023. The Author(s).)

Tuberculosis (TB) has been for many years a major public health problem since treatment is long and sometimes ineffective favoring the increase of multidrug-resistant mycobacteria (MDR-TB). Gene therapy is a novel and effective tool to regulate immune responses. In this study we evaluated the therapeutic effect of an adenoviral vector codifying osteopontin (AdOPN), a molecule known for their roles to favor Th1 and Th17 type-cytokine expression which are crucial in TB containment. A single dose of AdOPN administration in BALB/c mice suffering late progressive pulmonary MDR-TB produced significant lower bacterial load and pneumonia, due to higher expression of IFN-γ, IL-12, and IL-17 in coexistence with increase of granulomas in number and size, resulting in higher survival, in contrast with mice treated with the control adenovirus that codify the green fluorescent protein (AdGFP). Combined therapy of AdOPN with a regimen of second line antibiotics produced a better control of bacterial load in lung during the first days of treatment, suggesting that AdOPN can shorten chemotherapy. Taken together, gene therapy with AdOPN leads to higher immune responses against TB infection, resulting in a new potential treatment against pulmonary TB that can co-adjuvant chemotherapy.

Using intracellular-controlled photochemistry to track dynamic organelle processes is gaining attention due to its broad applications. However, most of the employed molecular probes usually require toxic photosensitizers and complex bioanalytical protocols. Here, the synthesis and performance of two new subcellular probes ( MitoT1 and MitoT2 ) are described. The probes undergo photooxidation in the damaged tissue of zebrafish, a model system for tissue regeneration studies. Using high-resolution confocal microscopy and fluorescence spectroscopy, we combine the mentioned photoinduced interconversion at the homeostatic membrane viscosity to track singlet oxygen activity selectively. The continuous and real-time biosensing method reported here provides a new approach for simultaneously detecting endogenous singlet oxygen and viscosity status.

Background: The Transforming Growth Factor β (TGFβ) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFβ family that participates in heart, liver and kidney development as revealed by the tgfbr3-null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish., Results: Here, we present CRISPR/Cas9-derived zebrafish Tgfbr3-null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3-null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFβ signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process., Conclusion: Betaglycan's "ligand presentation" function contributes to the optimal TGFβ signaling required for zebrafish chordacentra mineralization., (© 2021 American Association for Anatomy.)

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA 2 s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake ( Crotalus simus ) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

Morphological and behavioral evidence suggests that vasoinhibin is present in the central nervous system (CNS), triggering neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin reduces neuronal survival and differentiation of primary sensory neurons of the peripheral nervous system. To address the functional role played by vasoinhibin at the CNS, and to better understand the underlying mechanisms involved in its actions, we treated primary cultured hippocampal neurons obtained from embryonic day 16 (E16) mice with a human recombinant vasoinhibin. We examined the resulting cellular changes, focusing on neuronal cell death, and explored the local generation of vasoinhibin within the hippocampus. Our results show that vasoinhibin significantly reduced neuronal cell density and increased immunoreactive activated caspase-3 and TUNEL-positive staining at 72, 16, and 24 h, respectively. Furthermore, vasoinhibin increased the expression of proapoptotic genes BAX , BAD , BIM , and PUMA and decreased that of the antiapoptotic gene BCL-2 at 24 h, as assessed by quantitative real-time reverse transcription-polymerase chain reaction. Vasoinhibin effects were blocked by coincubation with a vasoinhibin antibody or with prolactin. Immunoreactive bands consistent with vasoinhibin were observed in hippocampal extracts by Western blot analysis, and a prolactin standard was cleaved to vasoinhibin by a hippocampal lysate in a heat- and cathepsin D inhibitor pepstatin A-dependent fashion. Taken together, these data support the notion that vasoinhibin is locally produced by cathepsin D within the embryonic mouse hippocampus, a brain region that plays a critical role in emotional regulation, resulting in decreased neuronal cell viability via the activation of the intrinsic apoptosis pathway.

Betaglycan (BG) and endoglin (ENG), homologous co-receptors of the TGF-β family, potentiate the signaling activity of TGF-β2 and inhibin A, and BMP-9 and BMP-10, respectively. BG exists as monomer and forms 1:1 growth factor (GF) complexes, while ENG exists as a dimer and forms 2:1 GF complexes. Herein, the structure of the BG orphan domain (BG O ) reveals an insertion that blocks the region that the endoglin orphan domain (ENG O ) uses to bind BMP-9, preventing it from binding in the same manner. Using binding studies with domain-deleted forms of TGF-β and BG O , as well as small-angle X-ray scattering data, BG O is shown to bind its cognate GF in an entirely different manner compared with ENG O . The alternative interfaces likely engender BG and ENG with the ability to selectively bind and target their cognate GFs in a unique temporal-spatial manner, without interfering with one another or other TGF-β family GFs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Small-molecule fluorescent probes having optimized optical properties, such as high photostability and brightness, local microenvironment sensitivity and specific subcellular localizations, are increasingly available. Although the basis for designing efficient fluorophores for bioimaging applications is well established, implementing an improvement in a given photophysical characteristic always tends to compromise another optical property. This problem has enormous consequences for in vivo imaging, where ensuring a specific localization and precise control of the probe response is challenging. Herein we discuss a fluorescent probe, CC334, as a case study of the chromenylium-cyanine family that commonly exhibits highly complex photophysical schemes and highly interfered bioanalytical responses. By an exhaustive and concise analysis of the CC334 optical responses including detailed spectroscopic calibrations, steady-state microenvironment effects, ultrafast photophysics analysis and computational studies, we elucidate a new strategy to apply the probe in the singlet oxygen reactive oxygen species (1O2-ROS) monitoring using in vitro and in vivo models. The probe provides a new avenue for designing fluorescent probes to understand the dynamic behavior of subcellular environments.

Background: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells., Methods: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting., Results: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells., Conclusions: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation., (© 2019 S. Karger AG, Basel.)

TGF-β type III receptor (TβRIII) is a co-receptor for TGFβ family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-βs, Bone Morphogenetic Proteins (BMP2/4) and Inhibins/Activins regulate different checkpoints during T cell differentiation. We have previously reported that TβRIII modulates T cell development by protecting developing thymocytes from apoptosis, however the role of this co-receptor in peripheral lymphocytes still remains elusive. Here we describe a detailed characterization of TβRIII expression in murine and human lymphocyte subpopulations demonstrating that this co-receptor is significantly expressed in T but not B lymphocytes and among them, preferentially expressed on naïve and central memory T cells. TβRIII was upregulated after TCR stimulation, in parallel to other early activation markers. In contrast, natural and induced Tregs downregulated TβRIII in association with FoxP3 upregulation. Finally, anti-TβRIII blocking experiments demonstrated that TβRIII promotes TGFβ-dependent iTreg conversion in vitro, and suggest that this co-receptor may be involved in modulating peripheral T cell tolerance and could be considered as a potential target to boost T cell immune responses., (Copyright © 2017. Published by Elsevier Inc.)

Breakdown of the blood-retinal barrier (BRB), as occurs in diabetic retinopathy and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing vision loss. Vasoinhibins are N-terminal fragments of prolactin that prevent BRB breakdown during diabetes. They modulate the expression of some transient receptor potential (TRP) family members, yet their role in regulating the TRP vanilloid subtype 4 (TRPV4) remains unknown. TRPV4 is a calcium-permeable channel involved in barrier permeability, which blockade has been shown to prevent and resolve pulmonary edema. We found TRPV4 expression in the endothelium and retinal pigment epithelium (RPE) components of the BRB, and that TRPV4-selective antagonists (RN-1734 and GSK2193874) resolve BRB breakdown in diabetic rats. Using human RPE (ARPE-19) cell monolayers and endothelial cell systems, we further observed that (i) GSK2193874 does not seem to contribute to the regulation of BRB and RPE permeability by vasoinhibins under diabetic or hyperglycemic-mimicking conditions, but that (ii) vasoinhibins can block TRPV4 to maintain BRB and endothelial permeability. Our results provide important insights into the pathogenesis of diabetic retinopathy that will further guide us toward rationally-guided new therapies: synergistic combination of selective TRPV4 blockers and vasoinhibins can be proposed to mitigate diabetes-evoked BRB breakdown.

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1-TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor-receptor contact.

Mycobacterium tuberculosis (Mtb) latent infection can lead to reactivation. The design of new strategies to prevent it is an important subject. B6D2F1 mice were infected intratracheally with a low dose of Mtb H37Rv to induce chronic infection. After 7 months, mice were treated with one dose of recombinant adenoviruses encoding TNFα, β defensin-3 and LL37. Immunosupression was induced 1 month later with corticosterone. In comparison with the control group, mice treated with adenoviruses showed significantly less bacterial load and pneumonia, the adenoviruses encoding TNFα and LL37 being the most efficient. Gene therapy based in a proinflammatory cytokine or antimicrobial peptides is a potentially useful system to prevent reactivation of latent tuberculosis., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-β receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125 I-TGF-β2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF-β) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF-β receptor type III, is a TGF-β coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF-β-binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time-lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583-603, 2015. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Transforming growth factor (TGF)-βs are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two TβRI:TβRII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF-β protomers was substituted to block receptor binding. The substituted dimer, TGF-β3 WD, bound the TβRII extracellular domain and recruited the TβRI with affinities indistinguishable from TGF-β3, but with one-half the stoichiometry. TGF-β3 WD was further shown to retain one-quarter to one-half the signalling activity of TGF-β3 in three established assays for TGF-β function. Single-molecule fluorescence imaging with GFP-tagged receptors demonstrated a measurable increase in the proportion of TβRI and TβRII dimers upon treatment with TGF-β3, but not with TGF-β3 WD. These results provide evidence that the two TβRI:TβRII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF-βs diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF-β superfamily that signal through a RI:RII:RII heterotrimer.

Betaglycan is a coreceptor for members of the transforming growth factor beta (TGF-beta) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Delta10 and Sol Delta11, bind TGF-beta, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-beta activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has K(d)'s in the low nanomolar range for the three TGF-beta isoforms, while those for Sol Delta10 and Sol Delta11 are 1-2 orders of magnitude higher. SPR analysis further shows that the K(d)'s of Sol Delta11 are not changed in the presence of Sol Delta10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-beta through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan's TGF-beta neutralizing activity.

We constructed recombinant adenoviruses encoding murine interferon-γ (AdIFNγ) and tested its therapeutic efficiency in a well characterized model of progressive pulmonary tuberculosis (TB) in Balb/c mice, infected through the trachea with the laboratory drug-susceptible H37Rv strain or multidrug-resistant (MDR) clinical isolate. When the disease was in a late phase, 2 months after infection, we administered by intratracheal cannulation a single dose [1.7 × 10 9 plaque forming units (pfu)] of AdIFNγ or the control adenovirus. Groups of mice were killed at different time-points and the lungs were examined to determine bacilli colony forming units (CFU), cytokine/chemokine gene expression, and CD4/CD8 subpopulations, and also subjected to automated histomorphometry. In comparison with the control group, after 2 weeks of treatment and during the next 6 months, AdIFNγ-treated animals infected with either the H37Rv strain or the MDR strain showed significantly lower bacilli loads and tissue damage (pneumonia), higher expressions of IFN-γ, tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS), and bigger granulomas. When compared with the results from conventional chemotherapy or AdIFNγ treatment alone, the combined treatment with AdIFNγ plus conventional chemotherapy shortened the time taken for reduction of bacillary load. This shows that gene therapy with AdIFNγ efficiently reconstituted the protective immune response and controlled the progress of pulmonary TB produced by MDR or non-MDR strains., (Copyright © 2008 The American Society of Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies.

The use of transgenic plants as new antigen-delivery systems for subunit vaccines has been increasingly explored. We herein report progress toward a papaya-based vaccine against cysticercosis. Synthetic peptides (KETc1, KETc12, KETc7) were successfully expressed in 19 different transgenic papaya clones and found to be immunogenic. Complete protection against cysticercosis was induced with the soluble extract of the clones that expressed the higher levels of transcripts in up to 90% of the immunized mice. This study represents a key step towards the development of a more effective, sustainable and affordable oral subunit vaccine against human and pig cysticercosis.

Transforming growth factor-beta (TGF-beta) is a key mediator in the pathogenesis of renal diseases. Betaglycan, also known as the type III TGF-beta receptor, regulates TGF-beta action by modulating its access to the type I and II receptors. Betaglycan potentiates TGF-beta; however, soluble betaglycan, which is produced by the shedding of the membrane-bound receptor, is a potent antagonist of TGF-beta. In the present work, we have used a recombinant form of soluble betaglycan (SBG) to prevent renal damage in genetically obese and diabetic db/db mice. Eight-wk-old db/db or nondiabetic (db/m) mice were injected intraperitoneally with 50 mug of SBG or vehicle alone three times a wk for 8 wk. The db/db mice that received vehicle presented albuminuria and increased serum creatinine, as well as glomerular mesangial matrix expansion. The db/db mice treated with SBG exhibited a reduction in serum creatinine, albuminuria, and structural renal damage. These effects were associated with lower kidney levels of mRNAs encoding TGF-beta1, TGF-beta2, TGF-beta3, collagen IV, collagen I, fibronectin, and serum glucocorticoid kinase as well as a reduction in the immunostaining of collagen IV and fibronectin. Our data indicate that SBG is a renoprotective agent that neutralized TGF-beta actions in this model of nephropathy. Because SBG has a high affinity for all TGF-beta isoforms, in particular TGF-beta2, it is found naturally in serum and tissues and its shedding may be regulated. We believe that SBG shall prove convenient for long-term treatment of kidney diseases and other pathologies in which TGF-beta plays a pathophysiological role.

The gonadal development of chicken embryo is regulated by hormones and growth factors. Transforming growth factor beta (TGF-beta) isoforms may play a critical role in the regulation of growth in chicken gonads. We have investigated the effect of the TGF-beta isoforms on the number of germ and somatic cells in the ovary of the chicken embryo. Ovaries were obtained from chicken embryos at 9 days of incubation. They were organ-cultured for 72 h in groups treated with TGF-beta1, TGF-beta2, soluble betaglycan, TGF-beta1 plus soluble betaglycan, or TGF-beta2 plus soluble betaglycan, and untreated (control). TGF-beta1 and TGF-beta2 diminished the somatic cell number in the ovary of the chicken embryo at this age by inhibiting the proliferation of the somatic cells without increasing apoptosis. On the other hand, TGF-beta1 and TGF-beta2 did not affect the number of germ cells in the cultured ovary. The capacity of TGF-beta1 and TGF-beta2 to diminish the number of somatic cells in the ovary was blocked with soluble betaglycan, a natural TGF-beta antagonist. However, changes in the location of germ cells within the ovary suggested that TGF-beta promoted the migration of the germ cells from the ovarian cortex to the medulla. Thus, TGF-beta affects germ and somatic cells in the ovary of the 9-day-old chicken embryo and inhibits the proliferation of somatic cells.

Transforming growth factor-beta (TGF-beta) and prostaglandins (PG) regulate the cell-mediated immune response, so it has been proposed that they affect the progression of pulmonary tuberculosis. Here we report that the administration of soluble betaglycan, a potent TGF-beta antagonist, and niflumic acid, a PG synthesis inhibitor, during the chronic phase of experimental murine tuberculosis enhanced Th1 and decreased Th2 cytokines, increased the expression of iNOS and reduced pulmonary inflammation, fibrosis and bacillary load. This immunotherapeutic approach resulted in significant control of the disease comparable to that achieved by anti-microbial treatment alone. Importantly, the combination of immunotherapy and anti-microbials resulted in an accelerated clearance of bacilli from the lung. These results confirm that TGF-beta and PG have a central pathophysiological role in the progression of pulmonary tuberculosis in the mouse and suggest that the addition of immunotherapy to conventional anti-microbial drugs might result in improved treatment of the disease.

TGFbeta1, beta2, and beta3 are 25kDa homodimeric polypeptides that play crucial non-overlapping roles in development, tumor suppression, and wound healing. They exhibit 70-82% sequence identity and transduce their signals by binding and bringing together the TGFbeta type I and type II receptors, TbetaRI and TbetaRII. TGFbeta2 differs from the other isoforms in that it binds TbetaRII weakly and is dependent upon the co-receptor betaglycan for function. To explore the physicochemical basis underlying these differences, we generated a series of single amino acid TbetaRII variants based on the crystal structure of the TbetaRII:TGFbeta3 complex and examined these in terms of their TGFbeta isoform binding affinity and their equilibrium stability. The results showed that TbetaRII Ile53 and Glu119, which contact TGFbeta3 Val92 and Arg25, respectively, together with TbetaRII Asp32, Glu55, and Glu75, which contact TGFbeta3 Arg94, each contribute significantly, between 1 kcal mol(-1) to 1.5 kcal mol(-1), to ligand binding affinities. These contacts likely underlie the estimated 4.1 kcal mol(-1) lower affinity with which TbetaRII binds TGFbeta2 as these three ligand residues are unchanged in TGFbeta1 but are conservatively substituted in TGFbeta2 (Lys25, Ile92, and Lys94). To test this hypothesis, a TGFbeta2 variant was generated in which these three residues were changed to those in TGFbetas 1 and 3. This variant exhibited receptor binding affinities comparable to those of TGFbetas 1 and 3. Together, these results show that these three residues underlie the lowered affinity of TGFbeta2 for TbetaRII and that all isoforms likely induce assembly of the TGFbeta signaling receptors in the same overall manner.

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.