Gd(III) and Yb(III) Complexes Derived from a New Water-Soluble Dioxopolyazacyclohexane Macrocycle.

A new macrocyclic ligand was synthesized by a reaction between diethylenetriaminepentaacetic (DTPA) dianhydride and trans-1,4-diaminocyclohexane, and the Gd(III) and Yb(III) complexes were prepared. The compounds were characterized by spectroscopic methods. Structural calculation by DFT shows that the amide linkages are arranged in such a way that a conformational strain is minimized in the macrocyclic frame. The coordination modes of the ligand and water in the metal complexes were also determined by DFT. The longitudinal relaxation time T1 was measured for aqueous solutions of the Gd(III) complex. The T1 relaxivity arises from the structural feature that a water molecule coordinated to the paramagnetic metal is surrounded by a large open space, through which the exchange of water occurs readily to shorten the relaxation time of water in the entire region, as a result of the chelate conformation defined strictly by the amide groups and the cyclohexane ring.

Green Metallic Nanoparticles for Cancer Therapy: Evaluation Models and Cancer Applications.

Metal-based nanoparticles are widely used to deliver bioactive molecules and drugs to improve cancer therapy. Several research works have highlighted the synthesis of gold and silver nanoparticles by green chemistry, using biological entities to minimize the use of solvents and control their physicochemical and biological properties. Recent advances in evaluating the anticancer effect of green biogenic Au and Ag nanoparticles are mainly focused on the use of conventional 2D cell culture and in vivo murine models that allow determination of the half-maximal inhibitory concentration, a critical parameter to move forward clinical trials. However, the interaction between nanoparticles and the tumor microenvironment is not yet fully understood. Therefore, it is necessary to develop more human-like evaluation models or to improve the existing ones for a better understanding of the molecular bases of cancer. This review provides recent advances in biosynthesized Au and Ag nanoparticles for seven of the most common and relevant cancers and their biological assessment. In addition, it provides a general idea of the in silico, in vitro, ex vivo, and in vivo models used for the anticancer evaluation of green biogenic metal-based nanoparticles.

LIN-35 beyond its classical roles: its function in the stress response.

The pocket protein family controls several cellular functions such as cell cycle, differentiation, and apoptosis, among others. However, its role in stress has been poorly explored. The roundworm Caenorhabditis elegans is a simple model organism whose genes are highly conserved during evolution. C. elegans has only one pocket protein, LIN-35; a retinoblastoma protein (pRB)-related protein similar to p130. To control the expression of some of its targets, LIN-35 interacts with E2F-DP (E2 transcription factor/dimerization partner complex) transcription factors and LIN-52, a member of SynMUV (Synthetic Muv) complex. Together, these proteins form the DRM complex, which is also known as the DREAM complex in mammals. In this review, we will focus on the role of LIN-35 and its partners in the stress response. It has been shown that LIN-35 is required to control starvation in L1 and L4 larval stages, and to induce starvation-induced germ apoptosis. Remarkably, during L1 starvation, insulin/IGF-1 receptor signaling (IIS), as well as the pathogenic, toxin, and oxidative stress-responsive genes, are repressed by LIN-35. The lack of lin-35 also triggers a downregulation of oxidative stress genes. Recent works showed that lin-35 and hpl-2 mutant animals showed enhanced resistance to UPRER. Additionally, hpl-2 mutant animals also exhibited upregulation of autophagic genes, suggesting that SynMuv/DRM proteins participate in this process. Finally, lin-35(n745) mutant animals overexpressed hsp-6, a chaperone that participated in the UPRmt. All of these data demonstrate that LIN-35 and its partners play an important role during the stress response.

Immune response of human cultured cells towards macrocyclic Fe2PO and Fe2PC bioactive cyclophane complexes.

Synthetic molecules that mimic the function of natural enzymes or molecules have untapped potential for use in the next generation of drugs. Cyclic compounds that contain aromatic rings are macrocyclic cyclophanes, and when they coordinate iron ions are of particular interest due to their antioxidant and biomimetic properties. However, little is known about the molecular responses at the cellular level. This study aims to evaluate the changes in immune gene expression in human cells exposed to the cyclophanes Fe2PO and Fe2PC. Confluent human embryonic kidney cells were exposed to either the cyclophane Fe2PO or Fe2PC before extraction of RNA. The expression of a panel of innate and adaptive immune genes was analyzed by quantitative real-time PCR. Evidence was found for an inflammatory response elicited by the cyclophane exposures. After 8 h of exposure, the cells increased the relative expression of inflammatory mediators such as interleukin 1; IRAK, which transduces signals between interleukin 1 receptors and the NFκB pathway; and the LPS pattern recognition receptor CD14. After 24 h of exposure, regulatory genes begin to counter the inflammation, as some genes involved in oxidative stress, apoptosis and non-inflammatory immune responses come into play. Both Fe2PO and Fe2PC induced similar immunogenetic changes in transcription profiles, but equal molar doses of Fe2PC resulted in more robust responses. These data suggest that further work in whole animal models may provide more insights into the extent of systemic physiological changes induced by these cyclophanes.

Insights Into the Hypometabolic Stage Caused by Prolonged Starvation in L4-Adult Caenorhabditis elegans Hermaphrodites.

Animals alter their reproductive cycles in response to changing nutritional conditions, to ensure that offspring production only occurs under favorable circumstances. These adaptive strategies include reversible hypometabolic states of dormancy such as "arrest" and "diapause." The free-living nematode Caenorhabditis elegans can arrest its life cycle during some larval stages without modifying its anatomy and physiology until conditions improve but it can also modify its morphological and physiological features to cope with harsh conditions and transition into diapause. The well-defined "dauer" diapause was described more than 40 years ago and has been the subject of comprehensive investigations. The existence of another hypometabolic state, termed adult reproductive diapause (ARD), has been debated after it was first described 10 years ago. Here, we review the current knowledge regarding the effect of food deprivation during the pre-reproductive larval and adult stages on overall organismal homeostasis, highlighting the implications on germ cell maintenance and fertility preservation.

Synthesis of Gold Nanoparticles Using Mimosa tenuiflora Extract, Assessments of Cytotoxicity, Cellular Uptake, and Catalysis.

Synthesis of gold nanoparticles (AuNPs) with plant extracts has gained great interest in the field of biomedicine due to its wide variety of health applications. In the present work, AuNPs were synthesized with Mimosa tenuiflora (Mt) bark extract at different metallic precursor concentrations. Mt extract was obtained by mixing the tree bark in ethanol-water. The antioxidant capacity of extract was evaluated using 2,2-diphenyl-1-picrylhydrazyl and total polyphenol assay. AuNPs were characterized by transmission electron microscopy, X-ray diffraction, UV-Vis and Fourier transform infrared spectroscopy, and X-ray photoelectron spectrometry for functional group determination onto their surface. AuMt (colloids formed by AuNPs and molecules of Mt) exhibit multiple shapes with sizes between 20 and 200 nm. AuMt were tested on methylene blue degradation in homogeneous catalysis adding sodium borohydride. The smallest NPs (AuMt1) have a degradation coefficient of 0.008/s and reach 50% degradation in 190s. Cell viability and cytotoxicity were evaluated in human umbilical vein endothelial cells (HUVEC), and a moderate cytotoxic effect at 24 and 48 h was found. However, toxicity does not behave in a dose-dependent manner. Cellular internalization of AuMt on HUVEC cells was analyzed by confocal laser scanning microscopy. For AuMt1, it can be observed that the material is dispersed into the cytoplasm, while in AuMt2, the material is concentrated in the nuclear periphery.

Caenorhabditis elegans respond to high-glucose diets through a network of stress-responsive transcription factors.

High-glycemic-index diets, as well as a sedentary lifestyle are considered as determinant factors for the development of obesity, type 2 diabetes, and cardiovascular diseases in humans. These diets have been shown to shorten the life span of C. elegans in a manner that is dependent on insulin signaling, but the participation of other signaling pathways have not been addressed. In this study, we have determined that worms fed with high-glucose diets show alterations in glucose content and uptake, triglyceride content, body size, number of eggs laid, egg-laying defects, and signs of oxidative stress and accelerated aging. Additionally, we analyzed the participation of different key regulators of carbohydrate and lipid metabolism, oxidative stress and longevity such as SKN-1/NRF2, HIF-1/HIF1α, SBP-1/SREBP, CRH-1/CREB, CEP-1/p53, and DAF-16/FOXO, in the reduction of lifespan in glucose-fed worms.

CTCF knockout reveals an essential role for this protein during the zebrafish development.

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

DPFF-1 transcription factor deficiency causes the aberrant activation of MPK-1 and meiotic defects in the Caenorhabditis elegans germline.

The d4 family of transcription factors consists of three members in mammals. DPF1/neuro-d4 is expressed mainly in neurons and the peripheral nervous system, and is important for brain development. DPF2/requiem/ubi-d4 is expressed ubiquitously and presumably functions as an apoptotic factor, especially during the deprivation of trophic factors. DPF3/cer-d4 is expressed in neurons and in the heart, and is important for heart development and function in zebrafish. In Drosophila, there is only one member, dd4, whose function is still unknown, but it is expressed in many tissues and is particularly abundant in the brain of developing embryos and in adults. Here, we present DPFF-1, the only member of this family of proteins in the nematode C. elegans. DPFF-1 is similar to its mammalian homolog DPF2/requiem/ubi-d4 because it is ubiquitously expressed during embryogenesis and in adult tissues, and because it is important for the induction of germ cell apoptosis during stress. Here, we show that dpff-1 null mutant animals produce less progeny than wild-type nematodes, presumably due to meiotic defects. Gonads of dpff-1 deficient animals showed more germ cells in pachytene and overexpressed the P-MPK-1 signal. Additionally, these animals presented higher levels of p53-induced germ cell apoptosis than wild-type animals. Furthermore, we observed that dpff-1 deficient animals are more sensitive to heat shock. This is the first report showing that the d4 family of transcription factors could be involved in meiosis and stress protection.

The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

The DEAD Box RNA helicase VBH-1 is a new player in the stress response in C. elegans.

For several years, DEAD box RNA helicase Vasa (DDX4) has been used as a bona fide germline marker in different organisms. C. elegans VBH-1 is a close homolog of the Vasa protein, which plays an important role in gametogenesis, germ cell survival and embryonic development. Here, we show that VBH-1 protects nematodes from heat shock and oxidative stress. Using the germline-defective mutant glp-4(bn2) we found that a potential somatic expression of vbh-1 might be important for stress survival. We also show that the VBH-1 paralog LAF-1 is important for stress survival, although this protein is not redundant with its counterpart. Furthermore, we observed that the mRNAs of the heat shock proteins hsp-1 and sip-1 are downregulated when vbh-1 or laf-1 are silenced. Previously, we reported that in C. elegans, VBH-1 was primarily expressed in P granules of germ cells and in the cytoplasm of all blastomeres. Here we show that during stress, VBH-1 co-localizes with CGH-1 in large aggregates in the gonad core and oocytes; however, VBH-1 aggregates do not overlap with CGH-1 foci in early embryos under the same conditions. These data demonstrate that, in addition to the previously described role for this protein in the germline, VBH-1 plays an important role during the stress response in C. elegans through the potential direct or indirect regulation of stress response mRNAs.

Bis{2-[2,5-bis-(pyridin-2-yl)-1H-imidazol-4-yl]pyridinium} tetra-cyanidoplatinate(II) tetra-hydrate.

The asymmetric unit of the title hydrated complex salt, (C18H14N5)2[Pt(CN)4]·4H2O, consists of one 2-[2,5-bis-(pyridin-2-yl)-1H-imidazol-4-yl]pyridinium cation, half a tetra-cyanidoplatinate(II) dianion, which is located about a crystallographic inversion center, and two water mol-ecules of crystallization. The Pt(II) atom has a square-planar coordination environment, with Pt-CCN distances of 1.992 (4) and 2.000 (4) Å. In the cation, there is an N-H⋯N hydrogen bond linking adjacent pyridinium and pyridine rings in positions 4 and 5. Despite this, the organic component is non-planar, as shown by the dihedral angles of 10.3 (2), 6.60 (19) and 15.66 (18)° between the planes of the central imidazole ring and the pyridine/pyridinium substituents in the 2-, 4- and 5-positions. In the crystal, cations and anions are linked via O-H⋯O, O-H⋯N and N-H⋯O hydrogen bonds, forming a three-dimensional network. Additional π-π, C-H⋯O and C-H⋯N contacts provide stabilization to the crystal lattice.

The zebrafish mutants for the V-ATPase subunits d, ac45, E, H and c and their variable pigment dilution phenotype.

BACKGROUND: The V-ATPase is a proton pump that creates an acidic medium, necessary for lysosome function and vesicular traffic. It is also essential for several developmental processes. Many enzymes, like the V-ATPase, are assemblies of multiple subunits, in which each one performs a specific function required to achieve full activity. In the zebrafish V-ATPase 15 different subunits form this multimeric complex and mutations in any of these subunits induce hypopigmentation or pigment dilution phenotype. We have previously found variability in the pigment dilution phenotype among five of the V-ATPase zebrafish mutants. This work presents additional information about such differences and is an update from a previous report. FINDINGS: We describe the variable phenotype severity observed among zebrafish V-ATPase pigment dilution mutants studying mRNA expression levels from their corresponding genes. At the same time we carried out phylogenetic analysis for this genes. CONCLUSIONS: Based in the similarities between different pigment dilution mutants we suggest that there is an essential role for V-ATPases in melanosome biogenesis and melanocyte survival. Neither variable expression levels for the different V-ATPase subunits studied here or the presence of duplicated genes seems to account for the variable phenotype severity from this group of mutants. We believe there are some similarities between the pigment dilution phenotype from zebrafish V-ATPase insertional mutants and pigment mutants obtained in a chemical screening ("Tubingen pigmentation mutants"). As for some of these "Tubingen mutants" the mutated gene has not been found we suggest that mutations in V-ATPase genes may be inducing their defects.

Germ cell survival in C. elegans and C. remanei is affected when the DEAD box RNA helicases VBH-1 or Cre-VBH-1 are silenced.

The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH-1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH-1 homolog in this gonochoristic species. We found that Cre-VBH-1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH-1 and Cre-VBH-1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre-vbh-1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh-1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre-vbh-1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH-1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one.

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans.

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (ChUP-1), [corrected] is involved in dietary cholesterol uptake in C. elegans. Animals lacking ChUP-1 [corrected] showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A ChUP-1-GFP [corrected] fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans ChUP-1. [corrected]. In-silico analysis identified two mammalian homologues of ChUP-1. [corrected]. Most interestingly, CRAC motifs are conserved in mammalian ChUP-1 [corrected] homologous. Our results suggest a role of ChUP-1 [corrected] in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.

Bioinformatic analysis of P granule-related proteins: insights into germ granule evolution in nematodes.

Germ cells in many animals possess a specialized cytoplasm in the form of granules that contain RNA and protein complexes essential for the function and preservation of the germline. The mechanism for the formation of these granules is still poorly understood; however, the lack of conservation in their components across different species suggests evolutionary convergence in the assembly process. Germ granules are assumed to be present in all nematodes with a preformed germline. However, few studies have clearly identified these structures in species other than Caenorhabditis elegans and even less have carried functional analysis to provide a broader panorama of the granules composition in the phylum. We adopted a bioinformatics approach to investigate the extension of conservation in nematodes of some known C. elegans germ granule components, as a proxy to understand germ granules evolution in this phylum. Unexpectedly, we found that, in nematodes, the DEAD box RNA helicase Vasa, a conserved protein among different phyla, shows a complex history of clade-specific duplications and sequence divergence. Our analyses suggest that, in nematodes, Vasa's function might be shared among proteins like LAF-1, VBH-1, and GLH-1/-2/-3 and GLH-4. Key components of P granules assembly in C. elegans, like the PGL protein family, are only preserved in Caenorhabditis species. Our analysis suggests that germ granules assembly may not be conserved in nematodes. Studies on these species could bring insight into the basic components required for this pathway.

Pigment dilution mutants from fish models with connection to lysosome-related organelles and vesicular traffic genes.

An interesting question in developmental biology is why mutations in genes with functions essential for the majority of cells produce diseases affecting only specific tissues. For example, pigment dilution disorders are often the consequence of mutations in conserved vesicular traffic genes. In Hermansky-Pudlak, Griscelli, and Chediak-Higashi pigment dilution syndromes, vesicular traffic mutations affect several organs with one characteristic in common: to carry out their functions they depend to a great extent on lysosome-related organelles (LROs), such as the melanosomes in melanocytes. Conserved multimeric complexes, present in most cell types, target proteins to lysosomes or selected LROs using transport vesicles. By studying these diseases or the model organisms that are defective in these processes, we have learned that every cell type possesses a unique way to regulate its vesicular traffic machinery and to assemble its multimeric complexes. This is accomplished by subunits from these multimeric complexes acting in a cell-specific manner. Here, we review several fish pigment dilution mutants that represent models for human vesicular traffic diseases.

Freeze-crack technique to study epidermal development in zebrafish using differential interference contrast microscopy and fluorescent markers.

The zebrafish is a model organism used to study organogenesis during vertebrate development; however epidermis development has been the focus of only a few studies. Thus, new methodologies to highlight and study epidermal cells could be valuable to deepen our understanding of skin development. Large-scale mutagenic screenings have already identified many zebrafish mutants, which are models for human developmental diseases, however only four epidermis mutants have been isolated. Novel screening techniques are needed to improve this collection. We designed and tested a novel freeze-crack technique to obtain, fix, and stain epidermal cells from 5 days postfertilization zebrafish larvae. Using commercially available fluorescent markers and differential interference contrast (DIC) microscopy, we were able to label and highlight subcellular structures such as microridges, cell boundaries, nuclei, and the Golgi complex from epidermis cells. Acquiring and processing epidermis samples from 15 to 75 larvae takes about 2-4 h, respectively. Therefore this method could be used as part of large-scale screenings. In addition, we present a more extensive protocol for antibody staining, which could be employed for more specific studies.