Binding studies of pyriproxyfen to DNA by multispectroscopic atomic force microscopy and molecular modeling methods

In this work, multispectroscopic atomic force microscopy and molecular modeling [ONIOM 2(B3LYP/6-31++G(d,p): Universal Force Field (UFF)) level] techniques were used to study the interaction between Calf-Thymus-DNA (CT-DNA) and pyriproxyfen (PYR) insecticide. The binding constant of PYR with double-strand deoxyribonucleic acid (ds-DNA) was obtained by ultraviolet-visible absorbance spectroscopy as 2.8×10(4) at 20°C. Thermodynamic parameters, that is, ΔH, ΔS°, and ΔG, were -53.82?kJ mol(-1), 96.11?J mol(-1), and -82.46?KJ mol(-1), respectively. Thermal denaturation study of DNA with PYR revealed the ΔT(m) of 3.0 and 6.0°C at r(i)=0.5 and 1.0, respectively. The Fourier transform infrared study showed a major interaction of PYR with G-C and A-T base pairs and a minor perturbation of the backbone PO(2) group. Further, PYR induces detectable changes in the circular dichroism spectrum of CT-DNA. In fluorimetric studies, the dynamic enhancement constants (k(D)) and bimolecular enhancement constant (k(B)) were calculated, which showed that the fluorescence enhancement was initiated by a static process in the ground state. The hybrid of quantum mechanical/molecular mechanics theoretical calculations revealed that the interaction is base sequence dependent, and PYR interacts more with DNA via the AT base sequence. From the data we concluded that PYR may interact with ds-DNA via two modes: intercalating and outside groove binding.     

In vitro study of DNA interaction with clodinafop-propargyl herbicide

The interaction of native calf thymus DNA with clodinafop-propargyl (CP), in 10 mM HEPES aqueous solutions at neutral pH 7.2, has been investigated by spectrophotometric, circular dichroism (CD), spectrofluorometric, melting temperature (Tm), and viscosimetric techniques. It was found that CP molecules could intercalate between base pairs of DNA as evidenced by hyperchromism in UV absorption band of DNA, an increase in melting temperature, a sharp increase in specific viscosity of DNA, induced CD spectral changes, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of CP, which indicates that it is able to release the intercalated MB completely. All results suggest that the CP interacts with calf thymus DNA by an intercalative mode of binding.     

Metabolomic analysis of cancer cachexia reveals distinct lipid and glucose alterations

Cancer cachexia remains a challenging clinical problem with complex pathophysiology and unreliable diagnostic tools. A blood test to detect this metabolic derangement would aid in early treatment of these patients. A ¹H NMR-based metabolomics approach was used to determine the unique metabolic fingerprint of cachexia and to search for biomarkers in serum samples taken from an established murine model of cancer cachexia. Male CD2F1 mice received a subcutaneous flank injection of C26 adenocarcinoma cells to induce experimental cancer-related cachexia. Two molecular markers of muscle atrophy, upregulation of the E3 ubiquitin ligase Muscle Ring Finger 1 (MuRF1) and aberrant glycosylation of β-dystroglycan (β-DG), were used to confirm muscle wasting in the tumor-bearing mice. Serum samples were collected for metabolomic analysis during the development of the cachexia: at baseline, when the tumor was palpable, and when the mice demonstrated cachexia. The unsupervised statistical analysis demonstrated a distinct metabolic profile with the onset of cachexia. The critical metabolic changes associated with cachexia included increased levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), with decreased serum glucose levels. Regression analysis demonstrated a very high correlation of the presence of aberrant glycosylation of β-DG with the unique metabolic profile of cachexia. This study demonstrates for the first time that metabolomics has potential as a diagnostic tool in cancer cachexia, and in further elucidating simultaneous metabolic pathway alterations due to this syndrome. In addition, variations in VLDL and LDL deserve more investigation as surrogate serum biomarkers for cancer cachexia.     

Experimental autoimmune encephalomyelitis (EAE) induced by antigen pulsed dendritic cells in the C57BL/6 mouse: influence of injection route.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) of the immune system, and are critically involved in initiation of immune responses in autoimmune diseases. They can modulate the nature of immune responses to stimulatory or tolerogenic fashion. Previous studies have demonstrated that the administration route of DCs is an important variable in eliciting anti-tumor immunity. In this study we used experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis to compare different protocols of DC delivery in autoimmunity or tolerance induction. Dendritic cells were generated from bone marrow cells of C57BL/6 mice by culturing in the presence of GM-CSF and IL-4 for 7 days, followed by 2 days culture with TNF-alpha. The obtained DCs were pulsed in vitro with myelin oligodendrocyte glycoprotein (MOG) peptide and injected (5 x 10(5) cells/mouse) via the intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route into female C57BL/6 mice. In some instances pertussis toxin was also injected zero and 48 hours after DC injection. After follow up of the mice pretreated in this way for 4 weeks, in the i.v. group in which no clinical signs of EAE occurred, the mice were immunized with MOG peptide for EAE induction via the common method and the results were compared with mice that were not pre-immunized. Only after three s.c. DC injections with pertussis toxin, the mice showed mild clinical signs of EAE, whereas mice given i.v. or i.p. injections with or without pertussis toxin failed to develop EAE after 4 weeks. Induction of EAE via the common method after three injections of TNF-alpha treated DCs, in i.v. injected groups showed no protection from EAE. It seems that several factors influence the tolerance versus immunity induction by DCs. Our results showed that the administration route of DCs is one of the pivotal factors in DC-based induction of autoimmune diseases.     

Potato starch synthases: Functions and relationships

Starch, a very compact form of glucose units, is the most abundant form of storage polyglucan in nature. The starch synthesis pathway is among the central biochemical pathways, however, our understanding of this important pathway regarding genetic elements controlling this pathway, is still insufficient. Starch biosynthesis requires the action of several enzymes. Soluble starch synthases (SSs) are a group of key players in starch biosynthesis which have proven their impact on different aspects of the starch biosynthesis and functionalities. These enzymes have been studied in different plant species and organs in detail, however, there seem to be key differences among species regarding their contributions to the starch synthesis. In this review, we consider an update on various SSs with an emphasis on potato SSs as a model for storage organs. The genetics and regulatory mechanisms of potato starch synthases will be highlighted. Different aspects of various isoforms of SSs are also discussed.     

A medium invasiveness multi-level patient’s specific template for pedicle screw placement in the scoliosis surgery

Background

Several methods including free-hand technique, fluoroscopic guidance, image-guided navigation, computer-assisted surgery system, robotic platform and patient’s specific templates are being used for pedicle screw placement. These methods have screw misplacements and are not always easy to be applied. Furthermore, it is necessary to expose completely a large portions of the spine in order to access fit entirely around the vertebrae.

Methods

In this study, a multi-level patient’s specific template with medium invasiveness was proposed for pedicle screw placement in the scoliosis surgery. It helps to solve the problems related to the soft tissues removal. After a computer tomography (CT) scan of the spine, the templates were designed based on surgical considerations. Each template was manufactured using three-dimensional printing technology under a semi-flexible post processing. The templates were placed on vertebras at four points—at the base of the superior-inferior articular processes on both left–right sides. This helps to obtain less invasive and more accurate procedure as well as true-stable and easy placement in a unique position. The accuracy of screw positions was confirmed by CT scan after screw placement.

Results

The result showed the correct alignment in pedicle screw placement. In addition, the template has been initially tested on a metal wire series Moulage (height 70 cm and material is PVC). The results demonstrated that it could be possible to implement it on a real patient.

Conclusions

The proposed template significantly reduced screw misplacements, increased stability, and decreased the sliding & the intervention invasiveness.

     

Detection and evaluation of non-recombinants in cDNA libraries by multiple cloning region PCR.

Bacteriophages that are routinely used in cDNA libraries do not require any biological selection for forming plaques. Thus parental non-recombinant phages are always found in variable proportions together with recombinant ones in all cDNA libraries. The presence of non-recombinants in significant proportions dilutes the abundance of rare cDNA species and makes library screening difficult. If the exact proportion of non-recombinants in a library were known, then one would screen proportionately more plaques to get a positive clone. In the absence of such information, screening is conventionally conducted on a number that is based on the titer of the library. We have devised a method using the flanking sequences from either side of the multiple cloning region (MCR) of all lambda phage vector derivatives as primers for PCR amplification. A non-recombinant phage produces a fragment equal to the size of the MCR, whereas a recombinant phage produces a fragment larger than the MCR, which is an MCR+ fragment. All cDNA libraries that we have studied show the presence of the MCR fragment (indicating non-recombinants) at variable proportions ranging between 6% and 36% of the total phages present. We also show that their presence negatively influences the retrieval of target cDNA sequences.     

Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon.

Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex, which associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the 2.0 A resolution crystal structure of the cytosolic part of pea Toc34 in complex with GDP and Mg2+. In the crystal, Toc34 molecules exist as dimers with features resembling those found in a small GTPase in complex with a GTPase activating protein (GAP). However, gel filtration experiments revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer solution at pH 7.2. Mutation of Arg 128, an essential residue for dimerization, to an Ala residue led to the formation of an exclusively monomeric species whose GTPase activity is significantly reduced compared to that of wild type Toc34. These results, together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer.