Synthesis, characterization, and structural studies of mercury(II) complexes of new bidentate phosphorus ylide

The reaction of Ph₂PCH₂CH₂PPh₂ (dppe) with BrCH₂C(O)C₆H₄NO₂ (1:1.05 molar ratio) in acetone produces a mixture of the new monophosphonium salt [Ph₂PCH₂CH₂PPh₂CH₂C(O)C₆H₄NO₂]Br (1) and the diphosphonium salt [NO₂C₆H₄C(O)CH₂PPh₂CH₂CH₂PPh₂CH₂C(O)C₆H₄NO₂]Br₂ (2). Compound 2 was insoluble in acetone and thus easily separated from the solution of 1. Further, by reacting both the mono- and diphosphonium salts with the appropriate bases the bidentate phosphorus ylides, [Ph₂PCH₂CH₂PPh₂CHC(O)C₆H₄NO₂] (3) and [NO₂C₆H₄C(O)CHPPh₂CH₂CH₂PPh₂CHC(O)C₆H₄NO₂] (4) were obtained. The reaction of ligand 3 with mercury(II) halides in dry methanol leads to the formation of the P,P-coordinated monomeric complexes {HgX₂(Ph₂PCH₂CH₂PPh₂CHC(O)C₆H₄NO₂)₂} [X = Cl (5), Br (6), I (7)]. The structure of complex 7 being unequivocally determined by single crystal X-ray diffraction techniques. Characterization of these species was also performed by elemental analysis, IR spectroscopy and ¹H, ³¹P, and ¹³C NMR techniques. These analyses being consistent with a 2:1 stoichiometry ylide/Hg(II) for compounds 5 through 7. Results obtained from theoretical studies are also consistent with a product in which two ylides are coordinated to the Hg(II) center through their phosphine groups, being this product the most stable among all the possible products.     

A robust universal method for extraction of genomic DNA from bacterial species

The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis analysis resulted in high A ₂₆₀/A ₂₈₀ ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale DNA isolation from various bacterial species.     

Simulation of DNA bases in water: Comparison of the Monte Carlo algorithm with molecular mechanics force fields

The interaction between the nucleic acid bases and solvent molecules has an important effect in various biochemical processes. We have calculated total energy and free energy of the solvation of DNA bases in water by Monte Carlo simulation. Adenine, guanine, cytosine, and thymine were first optimized in the gas phase and then placed in a cubic box of water. We have used the TIP3 model for water and OPLS for the nucleic acid bases. The canonical (T, V, N) ensemble at 25 degrees C and Metropolis sampling technique have been used. Good agreement with other available computational data was obtained. Radial distribution functions of water around each site of adenine, guanine, cytosine, and thymine have been computed and the results have shown the ability of the sites for hydrogen bonding and other interactions. The computations have shown that guanine has the highest value of solvation free energy and N7 and N6 in adenine and guanine, N3 in cytosine, and N3 and O4 in thymine have the largest radial distribution function. Monte Carlo simulation has also been performed using the CHARMM program under the same conditions, and the results of two procedures are compared.     

Desert hedgehog-patched 2 expression in peripheral nerves during Wallerian degeneration and regeneration

Hedgehog proteins are important in the development of the nervous system. As Desert hedgehog (Dhh) is involved in the development of peripheral nerves and is expressed in adult nerves, it may play a role in the maintenance of adult nerves and degeneration and regeneration after injury. We firstly investigated the Dhh-receptors, which are expressed in mouse adult nerves. The Dhh receptor patched(ptc)2 was detected in adult sciatic nerves using RT-PCR, however, ptc1 was undetectable under the same experimental condition. Using RT-PCR in purified cultures of mouse Schwann cells and fibroblasts, we found ptc2 mRNA in Schwann cells, and at much lower levels, in fibroblasts. By immunohistochemistry, Ptc2 protein was seen on unmyelinated nerve fibers. Then we induced crush injury to the sciatic nerves of wild-type (WT) and dhh-null mice and the distal stumps of injured nerves were analyzed morphologically at different time points and expression of dhh and related receptors was also measured by RT-PCR in WT mice. In dhh-null mice, degeneration of myelinated fibers was more severe than in WT mice. Furthermore, in regenerated nerves of dhh-null mice, minifascicular formation was even more extensive than in dhh-null intact nerves. Both dhh and ptc2 mRNA levels were down-regulated during the degenerative phase postinjury in WT mice, while levels rose again during the phase of nerve regeneration. These results suggest that the Dhh-Ptc2 signaling pathway may be involved in the maintenance of adult nerves and may be one of the factors that directly or indirectly determines the response of peripheral nerves to injury.     

The social context of cannibalism in migratory bands of the Mormon cricket

Cannibalism has been shown to be important to the collective motion of mass migratory bands of insects, such as locusts and Mormon crickets. These mobile groups consist of millions of individuals and are highly destructive to vegetation. Individuals move in response to attacks from approaching conspecifics and bite those ahead, resulting in further movement and encounters with others. Despite the importance of cannibalism, the way in which individuals make attack decisions and how the social context affects these cannibalistic interactions is unknown. This can be understood by examining the decisions made by individuals in response to others. We performed a field investigation which shows that adult Mormon crickets were more likely to approach and attack a stationary cricket that was side-on to the flow than either head- or abdomen-on, suggesting that individuals could reduce their risk of an attack by aligning with neighbours. We found strong social effects on cannibalistic behaviour: encounters lasted longer, were more likely to result in an attack, and attacks were more likely to be successful if other individuals were present around a stationary individual. This local aggregation appears to be driven by positive feedback whereby the presence of individuals attracts others, which can lead to further crowding. This work improves our understanding of the local social dynamics driving migratory band formation, maintenance and movement at the population level.     

Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (<Emphasis Type="Italic">Taxus baccata</Emphasis>)

Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.     

Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.     

ROS-mediated heme degradation and cytotoxicity induced by iron nanoparticles: hemoglobin and lymphocyte cells as targets

Nanoparticles (NPs) due to their small size and high surface area induce remarkable adverse effects on the biological systems. However, the exact mechanism by which NPs interacted with biological system and induce their adverse effects is still an enigma. Herein, the interaction of zero valent iron NPs (ZVFe NPs) with human hemoglobin (Hb) was evaluated using a variety of techniques including circular dichroism, fluorescence, and UV–visible (UV–vis) spectroscopy methods. Also, the cytotoxicity of ZVFe NPs on the human lymphocyte cell line as a model of blood system cell line was investigated by reactive oxygen species (ROS), caspase-9, and caspase-3 activities assays. It was revealed that ZVFe NP interaction resulted in heme displacement and degradation and induction of protein cabonylation. It was also shown that ZVFe NPs impaired the complexity of lymphocyte cells through ROS generation and apoptotic pathway. Together, these data suggest that NPs influence the biological system and induce adverse effects through ROS generation.     

Xenografting of testis tissue from bison calf donors into recipient mice as a strategy for salvaging genetic material

The objective was to evaluate the long-term outcome of testis tissue xenografting from neonatal bison calves as a model for closely related rare or endangered ungulates. Testis tissue was collected postmortem from two newborn bison calves (Bison bison bison) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 15 mice; eight fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo after grafting (for up to 16 mo). The retrieved xenografts were evaluated for seminiferous tubular density, tubular diameter, seminiferous tubular morphology, and identification of the most advanced germ cell type. Overall, 69% of the grafted testis fragments were recovered as xenografts. Xenografts weight increased (P < 0.02) approximately four-fold by 2 mo and 10-fold by 16 mo post-grafting. In testis xenografts, gradual maturational changes were evident, manifested as the first detection of the following at the times specified: seminiferous tubule expansion, 2 mo; spermatocytes, 6 mo; round spermatids, 12 mo; and elongated spermatids, 16 mo. Furthermore, there were differences between the two donor calves regarding the efficiency of spermatogenesis in xenografts. The timing of complete spermatogenesis approximately corresponded to the reported timing of sexual maturation in bison. This study demonstrated, apparently for the first time, that testis tissue xenografting from neonatal bison donors into recipient mice resulted in testicular maturation and complete development of spermatogenesis in the grafts.