Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (Kd = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (Kd = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (Kd = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (Kd = 1.6 μM) upon addition of Ni2+ (300 μM), possibly due to modest structural effect of Ni2+ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (Kd = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands. [Refer to PDF for graphical abstract

Temperatures during the dry cutting of titanium alloy using diamond composites with ceramic bonding phases

In this paper the thermal properties of diamond composites with ceramic bonding phases, such as the Ti–Si–C system with nanometric Ti(CN) and TiB₂ are presented. The thermal conductivities of the materials were analyzed by the laser pulse method. In addition, computational simulations of the temperature dependence on the distance from the cutting edge were performed according to the finite element method for the investigated composites, commercial PCD, and hypothetical diamond monocrystal.Досліджено теплові властивості алмазних композитів з керамічними зв’язуючими фазами, таких як системи Ti–Si–C з нанометровим Ti(CN) і TiB₂. Теплопровідності матеріалів проаналізовано за допомогою методу лазерного імпульсу. Крім того, методом скінченних елементів виконано числове моделювання залежності температури від відстані до ріжучої кромки для досліджених композитів, комерційного PCD і гіпотетичного монокристалу алмазу. Розглянуто дві швидкості різання в ході числового обчислення: 100 і 200 м/хв.Исследованы тепловые свойства алмазных композитов с керамическими связующими фазами, таких как системы Ti–Si–C с нанометровым Ti(CN) и TiB₂. Теплопроводности материалов проанализированы с помощью метода лазерного импульса. Кроме того, методом конечных элементов выполнено числовое моделирование зависимости температуры от расстояния до режущей кромки для исследованных композитов, коммерческого PCD и гипотетического монокристалла алмаза. Рассматривали две скорости резки в ходе числового вычисления: 100 и 200 м/мин

Dural tears in adult deformity surgery: Incidence, risk factors, and outcomes

Study Design: Retrospective cohort study. Objectives: Describe the rate of dural tears (DTs) in adult spinal deformity (ASD) surgery. Describe the risk factors for DT and the impact of this complication on clinical outcomes. Methods: Patients with ASD undergoing surgery between 2008 and 2014 were separated into DT and non-DT cohorts; demographics, operative details, radiographic, and clinical outcomes were compared. Statistical analysis included Results: A total of 564 patients were identified. The rate of DT was 10.8% (n = 61). Patients with DT were older (61.1 vs 56.5 years, Conclusions: The rate of DT was 10.8% in an ASD cohort. This is similar to rates of DT reported following surgery for degenerative pathology. A history of prior spine surgery, decompression, interbody fusion, and osteotomies are all associated with an increased risk of DT, but decompression is the only independent risk factor for DT

Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping

Many enzymes acting on DNA require Mg2+ ions not only for catalysis but also to bind DNA. Binding studies often employ Ca2+ as a substitute for Mg2+, to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca2+ mimics Mg2+, Ca2+ causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg2+ present as the DNA is cleaved so, to study the effect of Mg2+ on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca2+ or Mg2+ but, unlike wild-type SfiI with Ca2+, the binding was reversible. With both mutants, dissociation was slow with Ca2+ but was in one case much faster with Mg2+. Hence, Ca2+ can affect DNA binding differently from Mg2+. Moreover, SfiI is an archetypal system for DNA looping; on DNA with two recognition sites, it binds to both sites and loops out the intervening DNA. While the dynamics of looping cannot be measured with wild-type SfiI and Ca2+, it becomes accessible with the mutant and Mg2+

A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI)

Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5′-GGCGC/C-3′ (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction–modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins