Thyroid axis participates in high-temperature-induced male sex reversal through its activation by the stress response.

Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.

Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Stiffness of targeted layer-by-layer nanoparticles impacts elimination half-life, tumor accumulation, and tumor penetration.

Nanoparticle (NP) stiffness has been shown to significantly impact circulation time and biodistribution in anticancer drug delivery. In particular, the relationship between particle stiffness and tumor accumulation and penetration in vivo is an important phenomenon to consider in optimizing NP-mediated tumor delivery. Layer-by-layer (LbL) NPs represent a promising class of multifunctional nanoscale drug delivery carriers. However, there has been no demonstration of the versatility of LbL systems in coating systems with different stiffnesses, and little is known about the potential role of LbL NP stiffness in modulating in vivo particle trafficking, although NP modulus has been recently studied for its impact on pharmacokinetics. LbL nanotechnology enables NPs to be functionalized with uniform coatings possessing molecular tumor-targeting properties, independent of the NP core stiffness. Here, we report that the stiffness of LbL NPs is directly influenced by the mechanical properties of its underlying liposomal core, enabling the modulation and optimization of LbL NP stiffness while preserving LbL NP outer layer tumor-targeting and stealth properties. We demonstrate that the stiffness of LbL NPs has a direct impact on NP pharmacokinetics, organ and tumor accumulation, and tumor penetration-with compliant LbL NPs having longer elimination half-life, higher tumor accumulation, and higher tumor penetration. Our findings underscore the importance of NP stiffness as a design parameter in enhancing the delivery of LbL NP formulations.

Modification of Nanoparticles with Transferrin for Targeting Brain Tissues.

The delivery of therapeutics to brain tissues is one of the main challenges in neuropathology. For the past two decades, a variety of drug delivery systems has been designed to target components of the blood-brain barrier, including the transferrin receptor, a transmembrane glycoprotein highly expressed in the brain endothelium.In this protocol, we describe the use of transferrin protein to activate the surface of nanoparticles with the aim to direct their uptake in the brain. The molecule is bound by an amide linker to a PEGylated lipid commonly used in the preparation of lipid nanoparticles, micelles, and liposomes.

Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBCs). Because STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NPs) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-l-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to nontransformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated whether enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared with both unencapsulated cisplatin and cisplatin-loaded nontargeted NPs. In addition, because radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL NPs.

Synthesis of Doxorubicin and miRNA Stimuli-Sensitive Conjugates for Combination Therapy.

Recent advances in combination therapy by using chemotherapeutic drugs and small noncoding RNAs have highlighted the need for optimization of such agents to allow their carriage in a single delivery system. This protocol details the synthesis of a doxorubicin prodrug, where a NHS coupling reaction was used to sensitize the drug to the proteolytic activity of tumor microenvironments. The design of a lipid-modified miRNA by an S-S coupling reaction is also described. Modification of both, doxorubicin and miRNA, facilitated their simultaneous incorporation into mixed micelles for use in combination therapy against tumor cells.

The effect of low- and high-penetration light on localized cancer therapy.

The design of a delivery system allowing targeted and controlled drug release has been considered one of the main strategies used to provide individualized cancer therapy, to improve survival statistics, and to enhance quality-of-life. External stimuli including low- and high-penetration light have been shown to have the ability to turn drug delivery on and off in a non-invasive remotely-controlled fashion. The success of this approach has been closely related to the development of a variety of drug delivery systems - from photosensitive liposomes to gold nanocages - and relies on multiple mechanisms of drug release activation. In this review, we make reference to the two extremes of the light spectrum and their potential as triggers for the delivery of antitumor drugs, along with the most recent achievements in preclinical trials and the challenges to an efficient translation of this technology to the clinical setting.

Micelle-like nanoparticles as siRNA and miRNA carriers for cancer therapy.

Gene therapy has emerged as an alternative in the treatment of cancer, particularly in cases of resistance to chemo and radiotherapy. Different approaches to deliver genetic material to tumor tissues have been proposed, including the use of small non-coding RNAs due to their multiple mechanisms of action. However, such promise has shown limits in in vivo application related to RNA's biological instability and stimulation of immunity, urging the development of systems able to overcome those barriers. In this review, we discuss the use of RNA interference in cancer therapy with special attention to the role of siRNA and miRNA and to the challenges of their delivery in vivo. We introduce a promising class of drug delivery system known as micelle-like nanoparticles and explore their synthesis and advantages for gene therapy as well as the recent findings in in vitro, in vivo and clinical studies.

Applications of label-free, quantitative phase holographic imaging cytometry to the development of multi-specific nanoscale pharmaceutical formulations.

A label-free, high content, time-lapse holographic imaging system was applied to studies in pharmaceutical compound development. Multiple fields of cellular images are obtained over typically several day evaluations within standard CO2 incubators. Events are segmented to obtain population data of cellular features, which are displayed in scattergrams and histograms. Cell tracking is accomplished, accompanied by Cartesian plots of cell movement, as well as plots of cell features vs. time in novel 4-D displays of X position, Y position, time, and cell thickness. Our review of the instrument validation data includes 1) tracking of Giant HeLa cells, which may be undergoing neosis, a process of tumor stem cell generation; 2) tracking the effects of cell cycle related toxic agents on cell lines; 3) using MicroRNAs to reverse the polarization state in macrophages to induce tumor cell killing; 4) development of liposomal nanoformulations to overcome Multi-Drug Resistance (MDR) in ovarian cancer cells; and 5) development of dual sensitive micelles to specifically target matrix metalloproteinase 2 (MMP2) over-expressing cell lines. © 2017 International Society for Advancement of Cytometry.

Mixed Nanosized Polymeric Micelles as Promoter of Doxorubicin and miRNA-34a Co-Delivery Triggered by Dual Stimuli in Tumor Tissue.

Dual stimuli-sensitive mixed polymeric micelles (MM) are developed for co-delivery of the endogenous tumor suppressor miRNA-34a and the chemotherapeutic agent doxorubicin (Dox) into cancer cells. The novelty of the system resides in two stimuli-sensitive prodrugs, a matrix metalloproteinase 2 (MMP2)-sensitive Dox conjugate and a reducing agent (glutathione, GSH)-sensitive miRNA-34a conjugate, self-assembled in a single particle decorated with a polyethylene glycol corona for longevity, and a cell-penetrating peptide (TATp) for enhanced intracellular delivery. The MMP2-sensitivity of the system results in threefold higher cytotoxicity in MMP2-overexpressing HT1080 cells compared to low MMP2-expressing MCF7 cells. Cellular internalization of Dox increases by more than 70% after inclusion of TATp to the formulation. MMP2-sensitive MM also inhibits proliferation and migration of HT1080 cells. Moreover, GSH-sensitive MM allows for an efficient downregulation of Bcl2, survivin, and notch1 (65%, 55%, and 46%, respectively) in HT1080 cells. Combination of both conjugates in dual sensitive MM reduces HT1080 cell viability to 40% and expression of Bcl2 and survivin. Finally, 50% cell death is observed in 3D models of tumor mass. The results confirm the potential of the MM to codeliver miRNA-34a and doxorubicin triggered by dual stimuli inherent of tumor tissues.

siRNA Delivery by Stimuli-Sensitive Nanocarriers.

Since its discovery in the late 1990, small interfering RNA (siRNA) have quickly crept into the biopharmaceutical research as a new and powerful tool for the treatment of different human diseases based on altered gene-expression. Despite promising data from many pre-clinical studies, concrete hurdles still need to be overcome to bring therapeutic siRNAs in clinic. The design of stimuli-sensitive nanopreparations for gene therapy is a lively area of the current research. Compared to conventional systems for siRNA delivery, this type of platform can respond to local stimuli that are characteristics of the pathological area of interest, allowing the release of nucleic acids at the desired site. Acidic pH, de-regulated levels of enzymes, altered redox potential and magnetic field are examples of stimuli exploit to design stimuli-sensitive nanoparticles. In this review, we discuss on recent stimulisensitive strategies for siRNA delivery and we highlight on the potential of combining multiple stimuli-sensitive strategies in the same nano-platform for a better therapeutic outcome.

Are we accurately predicting bladder capacity in infants?

INTRODUCTION: Estimating bladder capacity is an important component in the evaluation of many urological disorders. For estimates to be of clinical value, precise reference ranges are needed. While accepted reference ranges have been established in adults and older children, none have been validated in infants. We endeavour to determine the normal bladder capacity of children less than 1 year of age. METHODS: We retrospectively reviewed the charts of children aged 0 to 12 months with cutaneous stigmata of spinal dysraphism who were referred to the urology clinic to rule out tethered cord between October 2004 and July 2011. Patients with normal urologic assessment, who did not have surgery during the time they were followed, were included in the study cohort. Urodynamic studies were performed using the Laborie Medical Technologies UDS-600. Bladder filling occurred via a catheter at a rate of 10% of the expected total bladder capacity/minute. Bladder capacity was defined as the volume of filling when the child voided around the catheter. We collected data, including age at urodynamics, bladder capacity, detrusor pressure at capacity, bladder compliance and length of follow-up. RESULT: In total, 46% (84/183) of patients had a normal urologic assessment and met the inclusion criteria. The median age was 9.0 months (interquartile range [IQR] 6.8-11.0). The average bladder capacity was 48.9 mL (standard deviation [SD] 32.8) and the mean detrusor pressure at capacity was 8.5 cmH2O (SD 10.0). Mean compliance was 14.1 mL/cmH2O (SD 13.6). The average length of follow-up was 40.7 months (SD 26.2) and during this interval no patients were found to have urologic or neurologic abnormalities and none underwent tethered cord release. CONCLUSION: Bladder capacity in infants with a median age of 9.0 months was found to be 48.9 mL. This is less than half of the volume predicted by a commonly employed formula. A novel method of estimating bladder capacity in infants is required.