Stapled Anoplin as an Antibacterial Agent

Anoplin is a linear 10-amino acid amphipathic peptide (Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2) derived from the venom sac of the solitary wasp. It has broad antimicrobial activity, including an antibacterial one. However, the inhibition of bacterial growth requires several dozen micromolar concentrations of this peptide. Anoplin is positively charged and directly interacts with anionic biological membranes forming an α-helix that disrupts the lipid bilayer. To improve the bactericidal properties of anoplin by stabilizing its helical structure, we designed and synthesized its analogs with hydrocarbon staples. The staple was introduced at two locations resulting in different charges and amphipathicity of the analogs. Circular dichroism studies showed that all modified anoplins adopted an α-helical conformation, both in the buffer and in the presence of membrane mimics. As the helicity of the stapled anoplins increased, their stability in trypsin solution improved. Using the propidium iodide uptake assay in Escherichia coli and Staphylococcus aureus, we confirmed the bacterial membrane disruption by the stapled anoplins. Next, we tested the antimicrobial activity of peptides on a range of Gram-negative and Gram-positive bacteria. Finally, we evaluated peptide hemolytic activity on sheep erythrocytes and cytotoxicity on human embryonic kidney 293 cells. All analogs showed higher antimicrobial activity than unmodified anoplin. Depending on the position of the staple, the peptides were more effective either against Gram-negative or Gram-positive bacteria. Anoplin[5-9], with a lower positive charge and increased hydrophobicity, had higher activity against Gram-positive bacteria but also showed hemolytic and destructive effects on eukaryotic cells. Contrary, anoplin[2-6] with a similar charge and amphipathicity as natural anoplin effectively killed Gram-negative bacteria, also pathogenic drug-resistant strains, without being hemolytic and toxic to eukaryotic cells. Our results showed that anoplin charge, amphipathicity, and location of hydrophobic residues affect the peptide destructive activity on the cell wall, and thus, its antibacterial activity. This means that by manipulating the charge and position of the staple in the sequence, one can manipulate the antimicrobial activity.

MAPK Inhibition Requires Active RAC1 Signaling to Effectively Improve Iodide Uptake by Thyroid Follicular Cells

The Sodium/Iodide Symporter (NIS) is responsible for the active transport of iodide into thyroid follicular cells. Differentiated thyroid carcinomas (DTCs) usually preserve the functional expression of NIS, allowing the use of radioactive iodine (RAI) as the treatment of choice for metastatic disease. However, a significant proportion of patients with advanced forms of TC become refractory to RAI therapy and no effective therapeutic alternatives are available. Impaired iodide uptake is mainly caused by the defective functional expression of NIS, and this has been associated with several pathways linked to malignant transformation. MAPK signaling has emerged as one of the main pathways implicated in thyroid tumorigenesis, and its overactivation has been associated with the downregulation of NIS expression. Thus, several strategies have been developed to target the MAPK pathway attempting to increase iodide uptake in refractory DTC. However, MAPK inhibitors have had only partial success in restoring NIS expression and, in most cases, it remained insufficient to allow effective treatment with RAI. In a previous work, we have shown that the activity of the small GTPase RAC1 has a positive impact on TSH-induced NIS expression and iodide uptake in thyroid cells. RAC1 is a downstream effector of NRAS, but not of BRAF. Therefore, we hypothesized that the positive regulation induced by RAC1 on NIS could be a relevant signaling cue in the mechanism underlying the differential response to MEK inhibitors, observed between NRAS- and BRAF-mutant tumors. In the present study, we found that the recovery of NIS expression induced through MAPK pathway inhibition can be enhanced by potentiating RAC1 activity in thyroid cell systems. The negative impact on NIS expression induced by the MAPK-activating alterations, NRAS Q61R and BRAF V600E, was partially reversed by the presence of the MEK 1/2 inhibitors AZD6244 and CH5126766. Notably, the inhibition of RAC1 signaling partially blocked the positive impact of MEK inhibition on NIS expression in NRAS Q61R cells. Conversely, the presence of active RAC1 considerably improved the rescue of NIS expression in BRAF V600E thyroid cells treated with MEK inhibitors. Overall, our data support an important role for RAC1 signaling in enhancing MAPK inhibition in the context of RAI therapy in DTC, opening new opportunities for therapeutic intervention.

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

The functional expression of the sodium–iodide symporter (NIS) at the membrane of differentiated thyroid cancer (DTC) cells is the cornerstone for the use of radioiodine (RAI) therapy in these malignancies. However, NIS gene expression is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment, which dramatically decreases patient survival. Several strategies have been attempted to increase the NIS mRNA levels in refractory DTC cells, so as to re-sensitize refractory tumors to RAI. However, there are many RAI-refractory DTCs in which the NIS mRNA and protein levels are relatively abundant but only reduced levels of iodide uptake are detected, suggesting a posttranslational failure in the delivery of NIS to the plasma membrane (PM), or an impaired residency at the PM. Because little is known about the molecules and pathways regulating NIS delivery to, and residency at, the PM of thyroid cells, we here employed an intact-cell labeling/immunoprecipitation methodology to selectively purify NIS-containing macromolecular complexes from the PM. Using mass spectrometry, we characterized and compared the composition of NIS PM complexes to that of NIS complexes isolated from whole cell (WC) lysates. Applying gene ontology analysis to the obtained MS data, we found that while both the PM-NIS and WC-NIS datasets had in common a considerable number of proteins involved in vesicle transport and protein trafficking, the NIS PM complexes were particularly enriched in proteins associated with the regulation of the actin cytoskeleton. Through a systematic validation of the detected interactions by co-immunoprecipitation and Western blot, followed by the biochemical and functional characterization of the contribution of each interactor to NIS PM residency and iodide uptake, we were able to identify a pathway by which the PM localization and function of NIS depends on its binding to SRC kinase, which leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals through PAK1 and PIP5K to promote ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. Besides providing novel insights into the regulation of NIS localization and function at the PM of TC cells, our results open new venues for therapeutic intervention in TC, namely the possibility of modulating abnormal SRC signaling in refractory TC from a proliferative/invasive effect to the re-sensitization of these tumors to RAI therapy by inducing NIS retention at the PM.

Characterization, Biological Activity, and Mechanism of Action of a Plant-Based Novel Antifungal Peptide, Cc-AFP1, Isolated From Carum carvi

Due to the increasing rate of invasive fungal infections and emerging antifungal resistance, development of novel antifungal drugs has been an urgent necessity. Antifungal peptides (AFPs) have recently attracted attention due to their unique ability to evade drug-resistant fungal pathogens. In this study, a novel AFP, Cc-AFP1, with a molecular weight of ~3.759 kDa, was isolated from Carum carvi L., purified by ammonium sulfate precipitation and reversed-phase HPLC and finally identified by sequence analysis using Edman degradation. Peptide sequence analysis revealed a fragment of 36 amino acid residues as RVCFRPVAPYLGVGVSGAVRDQIGVKLGSVYKGPRG for Cc-AFP1 with a net charge of +5 and a hydrophobicity ratio of 38%. The antifungal activity of Cc-AFP1 was confirmed against Aspergillus species with MIC values in the range of 8–16 µg/ml. Cc-AFP1 had less than 5% hemolytic activity at 8–16 µg/ml on human red blood cells with no obvious cytotoxicity against the HEK293 cell line. Stability analysis showed that the activity of Cc-AFP1 was maintained at different temperatures (20°C to 80°C) and pH (8 to 10). The results of a propidium iodide uptake and transmission electron microscopy showed that the antifungal activity of Cc-AFP1 could be attributed to alteration in the fungal cell membrane permeability. Taken together, these results indicate that Cc-AFP1 may be an attractive molecule to develop as a novel antifungal agent combating fungal infections cause by Aspergillus species.

Crosstalk Between Abnormal TSHR Signaling Activation and PTEN/PI3K in the Dedifferentiation of Thyroid Cancer Cells

Iodide uptake and the metabolism of thyroid cells are regulated by thyrotropin (TSH)-TSH receptor (TSHR) signaling. Thus, it is necessary to elevate serum TSH levels by T4 withdraw or rTSH administration to facilitate radioiodide (131I) therapy for differentiated thyroid cancer (DTC). However, non-iodide-avid metastases of DTC which is dedifferentiated do not respond to stimulation by high levels of TSH, suggesting abnormal TSH-TSHR signal transduction in cancer cells. In addition, PI3K/AKT/mTOR signaling activation has been shown to be associated with the dedifferentiated phenotype of thyroid cancer, but the mechanism remains elusive. Therefore, in this study, we aimed to explore the role of abnormal TSH-TSHR signaling activation in regulating iodide uptake and cell mobility in thyroid cancer and its relationship with PI3K/AKT/mTOR signaling. We found that in thyroid cancer cells, TSH binds TSHR coupled to the Gα12/13 protein and then activates RhoA through interacting with leukemia associated RhoA guanine exchange factor (LARG). This results in a promigration tumorigenic phenotype independent of canonical TSHR-GαS signaling that regulates the expression of molecules involved in iodine uptake and metabolism. We observed that signaling pathways downstream of Gα12/13 signaling were increased, while that of Gαs signaling was decreased in thyroid cancer cells undergoing dedifferentiation compared to control cells following stimulation with different levels of TSH. PI3K/AKT/mTOR signaling activation enhanced Gα12/13 signaling through increasing LARG levels but also inhibited the expression of molecules downstream of Gαs signaling, including thyroid-specific molecules, and iodide uptake. In summary, our results demonstrate the noncanonical activation of TSH-TSHR signaling and its role in increasing the cell mobility and dedifferentiation of thyroid cancer through crosstalk with PI3K/AKT/mTOR signaling.

Thyroid Hormones in the Human Body: A review

Thyroid gland is an essential endocrine gland that is present in the human body. This review highlights the production, regulation and disease conditions of the thyroid hormones. The thyroid gland is controlled with the help of hypothalamus and the pituitary gland that is present in the brain. The production of thyroid hormones involves five main steps such synthesis of thyroglobulin (TG), iodide uptake, iodination of thyroglobulin, storage and release. The two important hormones produced by the thyroid gland are Thyroxine (T4) and Triiodothyronine(T3). These two hormones are iodine containing derivatives. Deficiency of iodine in the body results in the reduction of T3 and T4 production. Excess and deficiency of these two hormones result in the major diseases like Hyper and Hypothyroidism. The synthetic forms of Thyroxine(T4) and Triiodothyronine(T3) used in the Pharmaceutical field are Levothyroxine and Liothyronine. Women are most commonly prone to these thyroid diseases comparatively to men. Undiagnosed disease conditions may become fatal. Levothyroxine therapy is commonly used for thyroidism which is a similar synthetic thyroid hormone. Also natural thyroid hormones that are obtained from the dried thyroid glands of animals are available. Thyroid function test and the measurement of T3, T4 plasma level becomes essential for the identification and regular maintenance of thyroid hormones in the body and human well-being.

Evaluation of potential toxicity of Steriplant©N aerosolization toward human alveolar cells A459 in vitro

Protection of patients against hospital-acquired infections is of major importance. Disinfection of magnetic resonance imaging suites is, due to their unique properties and environment particularly, difficult to implement. We developed an OPTI-JET CS MD 2ZE aerosolizator for disinfection of a magnetic resonance imaging suite using the electrolyzed oxidizing water biocide Steriplant©N. The disinfection of the magnetic resonance imaging suite with this system reduced from the number of colony formed unit/m3 air by 87% and 96% in 6 and 15 min of disinfection, respectively. It is well known that exposure of personnel or patients to aerosols may represent risk to the respiratory system; therefore, the aim of this study was to assess potential cytotoxicity and genotoxicity of Steriplant©N aerosolization toward human alveolar cells A459 in vitro . The A459 cells were exposed to aerosol containing different concentrations (50% and 100% v/v) of Steripalnt©N for 6 min in a chamber that had been constructed to simulate the conditions in the magnetic resonance imaging suite. The cytotoxicity was evaluated by measuring iodide uptake, and the genotoxicity was determined by measuring formation of phosphorylated H2AX histones, a marker for deoxyribonucleic acid double-strand breaks, immediately after the aerosolization and after 1, 4, and 24 h postincubation. The results demonstrated that aerosolization with Steriplant©N at conditions reflecting aerosolization in a magnetic resonance imaging suite is not cytotoxic and does not exhibit genotoxic potential in vitro.

The complex regulation of NIS expression and activity in thyroid and extrathyroidal tissues

The sodium iodide symporter (NIS) is an intrinsic plasma membrane protein that mediates active iodide transport into the thyroid gland and into several extrathyroidal tissues. NIS-mediated iodide uptake plays a pivotal role in the biosynthesis of thyroid hormones, of which iodide is an essential constituent. For 80 years radioiodide has been used for the diagnosis and treatment of thyroid cancer, a successful theranostic agent that is extending its use to extrathyroidal malignancies. The purpose of this review is to focus on the most recent findings regarding the mechanisms that regulate NIS both in thyroid and extra-thyroidal tissues. Among other issues, we discuss the different transcriptional regulatory elements that govern NIS transcription in different tissues, the epigenetic modifications that regulate its expression and the role that miRNAs play in fine tuning NIS after being transcribed. A review on the hormones, cytokines, and iodide itself that regulate NIS is provided. We also review the present stage of understanding NIS dysregulation in cancer, occupied mainly by convergent signaling pathways and by new insights in the route that NIS follows through different subcellular compartments to the plasma membrane. Furthermore, we cover NIS distribution and function in the increasing number of extrathyroidal tissues that express the symporter, as well as the role that NIS plays in tumor progression independently of its transport activity.