177Lu-Labeled Iron Oxide Nanoparticles Functionalized with Doxorubicin and Bevacizumab as Nanobrachytherapy Agents against Breast Cancer.

The use of conventional methods for the treatment of cancer, such as chemotherapy or radiotherapy, and approaches such as brachytherapy in conjunction with the unique properties of nanoparticles could enable the development of novel theranostic agents. The aim of our current study was to evaluate the potential of iron oxide nanoparticles, coated with alginic acid and polyethylene glycol, functionalized with the chemotherapeutic agent doxorubicin and the monoclonal antibody bevacizumab, to serve as a nanoradiopharmaceutical agent against breast cancer. Direct radiolabeling with the therapeutic isotope Lutetium-177 (177Lu) resulted in an additional therapeutic effect. Functionalization was accomplished at high percentages and radiolabeling was robust. The high cytotoxic effect of our radiolabeled and non-radiolabeled nanostructures was proven in vitro against five different breast cancer cell lines. The ex vivo biodistribution in tumor-bearing mice was investigated with three different ways of administration. The intratumoral administration of our functionalized radionanoconjugates showed high tumor accumulation and retention at the tumor site. Finally, our therapeutic efficacy study performed over a 50-day period against an aggressive triple-negative breast cancer cell line (4T1) demonstrated enhanced tumor growth retention, thus identifying the developed nanoparticles as a promising nanobrachytherapy agent against breast cancer.

Lineage-specific duplication of amphioxus retinoic acid degrading enzymes (CYP26) resulted in sub-functionalization of patterning and homeostatic roles.

BACKGROUND: During embryogenesis, tight regulation of retinoic acid (RA) availability is fundamental for normal development. In parallel to RA synthesis, a negative feedback loop controlled by RA catabolizing enzymes of the cytochrome P450 subfamily 26 (CYP26) is crucial. In vertebrates, the functions of the three CYP26 enzymes (CYP26A1, CYP26B1, and CYP26C1) have been well characterized. By contrast, outside vertebrates, little is known about CYP26 complements and their biological roles. In an effort to characterize the evolutionary diversification of RA catabolism, we studied the CYP26 genes of the cephalochordate amphioxus (Branchiostoma lanceolatum), a basal chordate with a vertebrate-like genome that has not undergone the massive, large-scale duplications of vertebrates. RESULTS: In the present study, we found that amphioxus also possess three CYP26 genes (CYP26-1, CYP26-2, and CYP26-3) that are clustered in the genome and originated by lineage-specific duplication. The amphioxus CYP26 cluster thus represents a useful model to assess adaptive evolutionary changes of the RA signaling system following gene duplication. The characterization of amphioxus CYP26 expression, function, and regulation by RA signaling demonstrated that, despite the independent origins of CYP26 duplicates in amphioxus and vertebrates, they convergently assume two main roles during development: RA-dependent patterning and protection against fluctuations of RA levels. Our analysis suggested that in amphioxus RA-dependent patterning is sustained by CYP26-2, while RA homeostasis is mediated by CYP26-1 and CYP26-3. Furthermore, comparisons of the regulatory regions of CYP26 genes of different bilaterian animals indicated that a CYP26-driven negative feedback system was present in the last common ancestor of deuterostomes, but not in that of bilaterians. CONCLUSIONS: Altogether, this work reveals the evolutionary origins of the RA-dependent regulation of CYP26 genes and highlights convergent functions for CYP26 enzymes that originated by independent duplication events, hence establishing a novel selective mechanism for the genomic retention of gene duplicates.

Exercise ventilatory parameters for the diagnosis of reactive pulmonary hypertension in patients with heart failure.

BACKGROUND: Reactive pulmonary hypertension (PH) in left heart disease is associated with poor prognosis. This study aimed to evaluate the diagnostic utility of exercise ventilatory parameters on cardiopulmonary exercise testing for the diagnosis of reactive PH in patients with heart failure (HF) and reduced ejection fraction. METHODS: This was a single-center, retrospective analysis of a prospectively collected database of 131 patients with HF who underwent in-hospital assessment for heart transplantation. Pulmonary hemodynamics was assessed by direct cardiac catheterization. Minute ventilation/carbon dioxide production (VE/VCO2) slope, partial pressure of end-tidal carbon dioxide (ETCO2) changes on exercise, oxygen pulse, and exercise oscillatory ventilation were determined from cardiopulmonary exercise testing. RESULTS: Sixty-one of 131 consecutive patients had reactive PH. VE/VCO2 slope (>41), change in ETCO2 on exercise (<1.2 mm Hg) and exercise oscillatory ventilation were independently associated with reactive PH. These 3 parameters in combination produced 3 possible diagnostic scenarios: (1) if all 3 criteria ("if all") were present, (2) if any 2 of the 3 criteria ("2 of 3") were present, and (3) if any of the criteria ("if any") were present. The corresponding positive/negative likelihood ratios for reactive PH if all 3 criteria were present were 3.73/0.83, if 2 of the 3 criteria were present were 2.19/0.45, and if any of the 3 criteria were present were 1.75/0.11. The posttest probability increased from 46% to 76% ("if all" present) and reduced to 9% (if none of the criteria was present). CONCLUSION: Ventilatory parameters on cardiopulmonary exercise test are associated with reactive PH in patients with HF. The absence of abnormalities in these 3 ventilatory parameters can effectively exclude reactive PH in patients with HF and poor ejection fraction.

Emerging technologies and challenges for better and safer drugs.

Regardless of stringent safety regulations and increased compound selectivity by pharmaceutical companies, prediction of toxicity in humans is still far from perfect and adverse drug reactions are still detected after drug marketing. High costs of failures due to toxicity has led pharmaceutical companies to search for screening methods that would allow detection of toxicity issues at an early stage and improve their preclinical and clinical toxicology. Thanks to the last decade's biotechnology revolution, new technologies like toxicogenomics have demonstrated the capacity to improve toxicity assessment. However, our understanding of toxicological mechanisms is still incomplete and a wide range of approaches must be used to gain insight into toxicity issues. Consequently, an array of in silico, in vitro and in vivo methods is utilized to predict toxicity and its causative mechanisms, improving drug development processes and minimizing costs of failure.

Automated cardiopulmonary resuscitation: a case study.

Rates of survival after cardiac arrest are low and correlate with the quality of cardiopulmonary resuscitation (CPR). Devices that deliver automated CPR (A-CPR) can provide sustained and effective chest compressions, which are especially useful during patient transfer and while simultaneous invasive procedures are being performed. The use of such devices can also release members of resuscitation teams for other work. This article presents a case study involving a man with acute myocardial infarction complicated by cardiogenic shock and pulmonary oedema. It describes how ED nursing and medical teams worked together to deliver A-CPR, discusses the use of A-CPR devices in a tertiary cardiac centre, and highlights the advantages of using such devices.

Vitamin A: a multifunctional tool for development.

Extensive research carried out over the last 100 years has established that the fat-soluble organic compound vitamin A plays crucial roles in early development, organogenesis, cell proliferation, differentiation and apoptosis as well as in tissue homeostasis. Given its importance during development, the delivery of vitamin A to the embryo is very tightly regulated with perturbations leading to severe malformations. This review discusses the roles of vitamin A during human development and the molecular mechanisms controlling its biological effects, hence bridging the gap between human development and molecular genetic work carried out in animal models. Vitamin A delivery during pregnancy and its developmental teratology in humans are thus discussed alongside work on model organisms, such as chicken or mice, revealing the molecular layout and functions of vitamin A metabolism and signaling. We conclude that, during development, vitamin A-derived signals are very tightly controlled in time and space and that this complex regulation is achieved by elaborate autoregulatory loops and by sophisticated interactions with other signaling cascades.

Iron oxide nanoflowers encapsulated in thermosensitive fluorescent liposomes for hyperthermia treatment of lung adenocarcinoma.

Magnetic hyperthermia (MHT) is in the spotlight of nanomedical research for the treatment of cancer employing magnetic iron oxide nanoparticles and their intrinsic capability for heat dissipation under an alternating magnetic field (AMF). Herein we focus on the synthesis of iron oxide nanoflowers (Nfs) of different sizes (15 and 35 nm) and coatings (bare, citrate, and Rhodamine B) while comparing their physicochemical and magnetothermal properties. We encapsulated colloidally stable citrate coated Nfs, of both sizes, in thermosensitive liposomes via extrusion, and RhB was loaded in the lipid bilayer. All formulations proved hemocompatible and cytocompatible. We found that 35 nm Nfs, at lower concentrations than 15 nm Nfs, served better as nanoheaters for magnetic hyperthermia applications. In vitro, magnetic hyperthermia results showed promising therapeutic and imaging potential for RhB loaded magnetoliposomes containing 35 nm Nfs against LLC and CULA cell lines of lung adenocarcinoma.

Bimetallic gold-platinum nanoparticles as a drug delivery system coated with a new drug to target glioblastoma.

A drug delivery nanosystem of noble bimetallic nanoparticles (NPs) which consists of Au NPs capped with Pt NPs (Au@Pt NPs) is constructed and functionalised with a quinazoline based small molecule (Au@Pt@Q NPs), acting as a theranostic agent against glioblastoma. Two different hydrothermal synthetic procedures for bimetallic Au@Pt NPs are presented and the resulting nanostructures are fully characterised by means of spectroscopic and microscopic methods. The imaging and targeting capacity of the new drug delivery system is assessed through fluorescent optical microscopy and cytotoxicity evaluations. The constructed Au@Pt NPs consist a monodispersed colloidal solution of 25 nm with photoluminescent, fluorescent and X-Ray absorption properties that confirm their diagnostic potential. Haemolysis testing demonstrated that Au@Pt NPs are biocompatible and fluorescent microscopy confirmed their entering the cells. Cytological evaluation of the NPs through MTT assay showed that they do not inhibit the proliferation of control cell line HEK293, whereas they are toxic in U87MG, U251 and D54 glioblastoma cell lines; rendering them selective targeting agents for treating glioblastoma.

Amphioxus spawning behavior in an artificial seawater facility.

Owing to its phylogenetic position at the base of the chordates, the cephalochordate amphioxus is an emerging model system carrying immense significance for understanding the evolution of vertebrate development. One important shortcoming of amphioxus as a model organism has been the unavailability of animal husbandry protocols to maintain amphioxus adults away from the field. Here, we present the first report of successful maintenance and spawning of Branchiostoma lanceolatum adults in a facility run on artificial seawater. B. lanceolatum has been chosen for this study because it is the only amphioxus species that can be induced to spawn. We provide a step-by-step guide for the assembly of such a facility and discuss the day-to-day operations required for successful animal husbandry of B. lanceolatum adults. This work also includes a detailed description of the B. lanceolatum spawning behavior in captivity. Our analysis shows that the induced spawning efficiency is not sex biased, but increases as the natural spawning season progresses. We find that a minor fraction of the animals undergo phases of spontaneous spawning in the tanks and that this behavior is not affected by the treatment used to induce spawning. Moreover, the induced spawning efficiency is not discernibly correlated with spontaneous spawning in the facility. Last, we describe a protocol for long-term cryopreservation of B. lanceolatum sperm. Taken together, this work represents an important step toward further establishing amphioxus as a laboratory animal making it more amenable to experimental research, and hence assists the coming of age of this emerging model.

From carrot to clinic: an overview of the retinoic acid signaling pathway.

Vitamin A is essential for the formation and maintenance of many body tissues. It is also important for embryonic growth and development and can act as a teratogen at critical periods of development. Retinoic acid (RA) is the biologically active form of vitamin A and its signaling is mediated by the RA and retinoid X receptors. In addition to its role as an important molecule during development, RA has also been implicated in clinical applications, both as a potential anti-tumor agent as well as for the treatment of skin diseases. This review presents an overview of how dietary retinoids are converted to RA, hence presenting the major players in RA metabolism and signaling, and highlights examples of treatment applications of retinoids. Moreover, we discuss the origin and diversification of the retinoid pathway, which are important factors for understanding the evolution of ligand-specificity among retinoid receptors.

Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation.

This chapter focuses on the in vitro biological evaluation of multisensitive nanocontainers as drug delivery systems for cancer treatment. Cancer tissues possess some unique characteristics such as increased temperature due to inflammation, thermal vulnerability (40-45 °C), low cellular pH, and redox instabilities. The employment of polymers bearing pH, thermo, and/or redox sensitivities in the synthesis of hollow polymeric nanostructures has led to the formulation of a variety of drug delivery vehicles that are capable of targeted delivery and trigger specific drug release. The cavity in the structure allows for the encapsulation of anticancer drugs as well as other moieties with anticancer activity, like iron oxide magnetic nanoparticles. The drug loading and release capability of the nanocontainers is evaluated prior to biological studies in order to determine the concentration of the drug in the structure. The in vitro assessment includes cytotoxicity studies, quantitatively through the colorimetric MTT assay as well as qualitatively via the scratch-wound healing assay, on both cancer and healthy cell lines. The cellular localization of the studied drug-loaded and unloaded nanocontainers is determined through confocal fluorescence microscopy.

Gold nanoparticle decorated pH-sensitive polymeric nanocontainers as a potential theranostic agent.

A pH-sensitive system of hollow P(MAA-co-MBA-co-AA) nanocontainers (NCs) modified with gold nanoparticles (GNCs) has been developed for theranostic applications, drug delivery and real time monitoring through imaging. The GNCs were synthesised by the distillation precipitation copolymerization procedure followed by in situ synthesis and embodiment of gold nanoparticles on the polymeric matrix (CSNs). Separately, citrate capped gold nanoparticles (GNPs) were also synthesized and compared with the GNCs for their fluorescence and cellular localization ability. The GNCs were tested for their drug loading and release behavior in response to the anticancer drug doxorubicin (DOX) at different pH values. Sustained drug release was observed at acidic pH. The viability of MCF-7 breast cancer cells and HEK-293 human embryonic kidney cells in relation to the GNCs, GNPs, GNC@DOX and DOX was also evaluated. GNCs and GNPs at the tested concentrations did not inhibit proliferation at either cell lines, whereas the GNCs@DOX presented comparable results. Cellular migration of MCF-7 cells treated with GNCs or GNPs was evaluated through the scratch-wound healing assay but no significant inhibition was detected. GNCs' and GNPs' fluorescence ability was exploited for assessing cellular localization through confocal laser scanning microscopy. GNCs after only 1 h of treatment were found in the cytoplasm of MCF7 cells, whereas GNCs@DOX were localized in the nucleus; the desirable site of action of DOX.

Sudden hearing loss in a family with GJB2 related progressive deafness.

Mutations of GJB2, the gene encoding connexin 26, have been associated with prelingual, sensorineural hearing loss of mild to profound severity. One specific mutation, the 35delG, has accounted for the majority of mutations detected in the GJB2 gene in Caucasian populations. Recent studies have described progression of hearing loss in a proportion of cases with GJB2 deafness. We report an unusual family with four 35delG homozygous members, in which the parents were deaf-mute whilst both children had a postlingual progressive hearing loss. Furthermore, the son suffered from sudden hearing loss.

Isolation and characterization of axolotl NPDC-1 and its effects on retinoic acid receptor signaling.

Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of retinoic acid receptor (RAR)-mediated transcription. To obtain comparative and developmental insights about NPDC-1 function, we cloned the axolotl (Ambystoma mexicanum) orthologue and measured transcript abundances among tissues sampled during the embryonic and juvenile phases of development, and also during spinal cord regeneration. Structurally, the axolotl orthologue of NPDC-1 retained sequence identity to mammalian sequences in all functional domains. Functionally, we observed that axolotl NPDC-1 mRNA expression peaked late in embryogenesis, with highest levels of expression occurring during the time of limb development, a process regulated by retinoic acid signaling. Also similar to what has been observed in mammals, axolotl NPDC-1 directly interacts with axolotl RAR, modulates axolotl RAR DNA binding, and represses cell proliferation and axolotl RAR-mediated gene transcription. These data justify axolotl as a model to further investigate NPDC-1 and its role in regulating retinoic acid signaling.

Roles of retinoic acid and Tbx1/10 in pharyngeal segmentation: amphioxus and the ancestral chordate condition.

BACKGROUND: Although chordates descend from a segmented ancestor, the evolution of head segmentation has been very controversial for over 150 years. Chordates generally possess a segmented pharynx, but even though anatomical evidence and gene expression analyses suggest homologies between the pharyngeal apparatus of invertebrate chordates, such as the cephalochordate amphioxus, and vertebrates, these homologies remain contested. We, therefore, decided to study the evolution of the chordate head by examining the molecular mechanisms underlying pharyngeal morphogenesis in amphioxus, an animal lacking definitive neural crest. RESULTS: Focusing on the role of retinoic acid (RA) in post-gastrulation pharyngeal morphogenesis, we found that during gastrulation, RA signaling in the endoderm is required for defining pharyngeal and non-pharyngeal domains and that this process involves active degradation of RA anteriorly in the embryo. Subsequent extension of the pharyngeal territory depends on the creation of a low RA environment and is coupled to body elongation. RA further functions in pharyngeal segmentation in a regulatory network involving the mutual inhibition of RA- and Tbx1/10-dependent signaling. CONCLUSIONS: These results indicate that the involvement of RA signaling and its interactions with Tbx1/10 in head segmentation preceded the evolution of neural crest and were thus likely present in the ancestral chordate. Furthermore, developmental comparisons between different deuterostome models suggest that the genetic mechanisms for pharyngeal segmentation are evolutionary ancient and very likely predate the origin of chordates.

NPDC-1, a novel regulator of neuronal proliferation, is degraded by the ubiquitin/proteasome system through a PEST degradation motif.

Neural proliferation and differentiation control protein-1 (NPDC-1) is a protein expressed primarily in brain and lung and whose expression can be correlated with the regulation of cellular proliferation and differentiation. Embryonic differentiation in brain and lung has classically been linked to retinoid signaling, and we have recently characterized NPDC-1 as a regulator of retinoic acid-mediated events. Regulators of differentiation and development are themselves highly regulated and usually through multiple mechanisms. One such mechanism, protein degradation via the ubiquitin/proteasome degradation pathway, has been linked to the expression of a number of proteins involved in control of proliferation or differentiation, including cyclin D1 and E2F-1. The data presented here demonstrate that NPDC-1 is likewise degraded by the ubiquitin/proteasome system. MG-132, a proteasome inhibitor, stabilized the expression of NPDC-1 and allowed detection of ubiquitinated NPDC-1 in vivo. A PEST motif (rich in proline, glutamine, serine, and threonine) located in the carboxyl terminus of NPDC-1 was shown to target the protein for degradation. Deletion of the PEST motif increased NPDC-1 protein stability and NPDC-1 inhibitory effect on retinoic acid-mediated transcription. NPDC-1 was phosphorylated by several kinases, including extracellular signal-regulated kinase. Phosphorylation of NPDC-1 increased the in vitro rate of NPDC-1 ubiquitination. The MEK inhibitor, PD-98059, an inhibitor of extracellular signal-regulated activation, also inhibited the formation of ubiquitinated NPDC-1 in vivo. Together these results suggest that retinoic acid signaling can be modulated by the presence of NPDC-1 and that the protein level and activity of NPDC-1 can be regulated by phosphorylation-mediated proteasomal degradation.

A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.