Structural determination of arginine-linked cisplatin complexes via IRMPD action spectroscopy: arginine binds to platinum via NO- binding mode.

Cisplatin, (NH3)2PtCl2, has been known as a successful metal-based anticancer drug for more than half a century. Its analogue, Argplatin, arginine-linked cisplatin, (Arg)PtCl2, is being investigated because it exhibits reactivity towards DNA and RNA that differs from that of cisplatin. In order to understand the basis for its altered reactivity, the deprotonated and sodium cationized forms of Argplatin, [(Arg-H)PtCl2]- and [(Arg)PtCl2 + Na]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy in the IR fingerprint and hydrogen-stretching regions. Complementary electronic structure calculations are performed using density functional theory approaches to characterize the stable structures of these complexes and to predict their infrared spectra. Comparison of the theoretical IR spectra predicted for various stable conformations of these Argplatin complexes to their measured IRMPD spectra enables determination of the binding mode(s) of Arg to the Pt metal center to be identified. Arginine is found to bind to Pt in a bidentate fashion to the backbone amino nitrogen and carboxylate oxygen atoms in both the [(Arg-H)PtCl2]- and [(Arg)PtCl2 + Na]+ complexes, the NO- binding mode. The neutral side chain of Arg also interacts with the Pt center to achieve additional stabilization in the [(Arg-H)PtCl2]- complex. In contrast, Na+ binds to both chlorido ligands in the [(Arg)PtCl2 + Na]+ complex and the protonated side chain of Arg is stabilized via hydrogen-bonding interactions with the carboxylate moiety. These findings are consistent with condensed-phase results, indicating that the NO- binding mode of arginine to Pt is preserved in the electrospray ionization process even under variable pH and ionic strength.

Metallic powder-bed based 3D printing of cellular scaffolds for orthopaedic implants: A state-of-the-art review on manufacturing, topological design, mechanical properties and biocompatibility.

Metallic cellular scaffold is one of the best choices for orthopaedic implants as a replacement of human body parts, which could improve life quality and increase longevity for the people needed. Unlike conventional methods of making cellular scaffolds, three-dimensional (3D) printing or additive manufacturing opens up new possibilities to fabricate those customisable intricate designs with highly interconnected pores. In the past decade, metallic powder-bed based 3D printing methods emerged and the techniques are becoming increasingly mature recently, where selective laser melting (SLM) and selective electron beam melting (SEBM) are the two representatives. Due to the advantages of good dimensional accuracy, high build resolution, clean build environment, saving materials, high customisability, etc., SLM and SEBM show huge potential in direct customisable manufacturing of metallic cellular scaffolds for orthopaedic implants. Ti-6Al-4V to date is still considered to be the optimal materials for producing orthopaedic implants due to its best combination of biocompatibility, corrosion resistance and mechanical properties. This paper presents a state-of-the-art overview mainly on manufacturing, topological design, mechanical properties and biocompatibility of cellular Ti-6Al-4V scaffolds via SLM and SEBM methods. Current manufacturing limitations, topological shortcomings, uncertainty of biocompatible test were sufficiently discussed herein. Future perspectives and recommendations were given at the end.

Evaluation of Hybrid Theoretical Approaches for Structural Determination of a Glycine-Linked Cisplatin Derivative via Infrared Multiple Photon Dissociation (IRMPD) Action Spectroscopy.

To gain a better understanding of the binding mechanism and assist in the optimization of chemical probing and drug design applications, experimental and theoretical studies of a series of amino acid-linked cisplatin derivatives are being pursued. Glyplatin (glycine-linked cisplatin) was chosen for its structural simplicity and to enable backbone effects to be separated from side-chain effects on the structure and reactivity of ornithine- and lysine-linked cisplatin (Ornplatin and Lysplatin, respectively). Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments were performed on Glyplatin to characterize its structure and guide the selection of the most effective hybrid theoretical approach for determining its structure and IR spectrum. The simplicity of the Glyplatin system allows a wide variety of density functionals, treatments of the Pt center including the use of all-electron basis sets vs valence basis sets combined with an effective core potential (ECP), and basis sets for all other atoms to be evaluated at a reasonable computational cost. The results for Glyplatin provide the foundation for calculations of more complex amino acid-linked cisplatin derivatives such as Ornplatin and Lysplatin. Present results suggest that the B3LYP/mDZP/def2-TZVP hybrid method can be effectively employed for structural and IR characterization of more complex amino acid-linked cisplatin complexes and their nucleic acid derivatives.

Local review of treatment of hand enchondroma (artificial bone substitute versus autologous bone graft) in a tertiary referral centre: 13 years' experience.

OBJECTIVE: To evaluate the treatment outcomes of enchondroma of the hand with artificial bone substitute versus autologous (iliac) bone graft. DESIGN: Historical cohort study. SETTING: Tertiary referral centre, Hong Kong. PATIENTS: A total of 24 patients with hand enchondroma from January 2001 to December 2013 who underwent operation at the Prince of Wales Hospital and Alice Ho Miu Ling Nethersole Hospital in Hong Kong were reviewed. Thorough curettage of the tumour was performed in all patients, followed by either autologous bone graft impaction under general anaesthesia in 13 patients, or artificial bone substitute in 11 patients (10 procedures were performed under local or regional anaesthesia and 1 was done under general anaesthesia). The functional outcomes and bone incorporation were measured by QuickDASH (shortened version of the Disabilities of the Arm, Shoulder and Hand questionnaire) scores and radiological appearance, respectively. The mean follow-up period was 59 months. RESULTS: There were eight men and 16 women, with a mean age of 40 years. Overall, 17 cases involved phalangeal bones and seven involved metacarpal bones. Among both groups of patients, most of the affected digits had good range of motion and function after surgery. One patient in each study group had complications of local soft tissue inflammation. One patient in the artificial bone substitute group was suspected to have recurrence 8 years after operation. Among the autologous bone graft group, four patients had persistent donor site morbidity at the last follow-up. In all patients, radiographs showed satisfactory bone incorporation. CONCLUSIONS: Artificial bone substitute is a safe and effective treatment option for hand enchondroma, with satisfactory functional and radiographic outcomes. Artificial bone substitute offers the additional benefits of enabling the procedure to be done under local anaesthesia on a day-case basis with minimal complications.

Reconstruction of hypoplastic thumb using hemi-longitudinal metatarsal transfer.

The treatment of hypoplastic thumb (modified Blauth's type IIIb and IV) by pollicization is culturally unfavourable in the Chinese population and digit preservation is preferred. An innovative reconstruction method using a nonvascularized hemi-longitudinal metatarsal graft was performed in six cases with an average follow-up of 87.7 months. Overall hand function was good, as assessed using the Jebsen hand function test. Grip strength and pinch power were significantly weaker than the normal contralateral hand. There was no neurovascular or wound complication. The only donor site complication was a metatarsal fracture, which healed uneventfully with casting. There had been no permanent morbidity to the donor site, as all donor metatarsals hypertrophied and regained normal growth potentials. Linear growth of the transferred metatarsals was evident radiologically (average 1.5 mm/year). Free hemi-longitudinal metatarsal transfer is a feasible method with good functional outcome in the attainment of a 5-digit hand in patients with type IIIb/IV hypoplastic thumb.

Laparoscopy: an excellent tool in the management of primary omental torsion in children.

INTRODUCTION: Primary omental torsion (POT) is an uncommon acute condition, often occurring in obese children. The clinical presentation usually mimics that of acute appendicitis, and preoperative radiologic imaging may not be helpful in the diagnosis. In this paper, we report our experience of using laparoscopy in diagnosing and treating POT in children. MATERIALS AND METHODS: A retrospective review of all cases of POT from 1998 to 2006 was performed. The efficacy and safety of using the laparoscope in the management of omental torsion was assessed. RESULTS: There were 5 boys with a mean age of 8.8 years (range, 5-11) included in the study. The majority of the patients were overweight, and all presented with abdominal pain without other gastrointestinal symptoms. In all patients, there was marked localized tenderness at the right side but without rebound tenderness or guarding. Preoperative investigations did not help in the diagnosis in all cases. A laparoscopic examination was performed and, in all cases, the diagnosis of POT was accurately made. The omentum was either adherent to the anterior abdominal wall or to the ascending colon. In all cases, the twisted omentum was successfully removed by the laparoscopic technique. Rapid recovery was universally observed, with the disappearance of pain and a rapid resumption of diet and discharge from the hospital on the first postoperative day. Histology showed gangrenous omentum in all cases. All the patients were well and had good cosmetic results on the follow-up. CONCLUSIONS: Laparoscopy is an excellent tool for both diagnosing and treating omental torsion in children.

Is symptomatology useful in distinguishing between carpal tunnel syndrome and cervical spondylosis?

Hand paraesthesia is a common symptom found in patients either with carpal tunnel syndrome or cervical spondylosis. To differentiate between the two conditions, it is important to identify additional diagnostic symptoms. Ninety-two patients with operated carpal tunnel syndrome and 138 patients with spinal surgery for cervical spondylosis were reviewed. After exclusion of cases co-morbid with both cervical spondylosis and carpal tunnel syndrome or other neurological disorders, 44 patients with carpal tunnel syndrome and 41 patients with cervical spondylosis were compared. There were significant differences in the symptomatology between the two groups. In carpal tunnel syndrome, 84% had nocturnal paraesthesia, 82% hand paraesthesia were aggravated by hand activity, and hand pain occurred in 64%. The incidences were only 10%, 7% and 10%, respectively in cervical spondylosis. Neck pain was present in 76% of cervical spondylosis but only in 14% of carpal tunnel syndrome, and lower limb symptoms were present in 44% of cervical spondylosis and only 9% in carpal tunnel syndrome.

Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches.

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 microM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA.

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Structural and kinetic studies of a cisplatin-modified DNA icosamer binding to HMG1 domain B.

The high mobility group (HMG) domain is a DNA-binding motif found in the non-histone chromosomal proteins, HMG1 and HMG2, and some transcription factors. Experimental evidence has demonstrated that HMG-domain proteins can play a role in sensitizing cells to the anticancer drug cisplatin. Fluorescence resonance energy transfer (FRET) experiments were performed in the present study to investigate structural changes that accompany complex formation between the HMG domain B of HMG1 and a cisplatin-modified, 20-base pair double-stranded DNA probe containing fluorescein and rhodamine tethered at its two ends. The binding affinity of HMG1 domain B for the cisplatin-modified DNA probe was investigated in fluorescence titration experiments, and a value of 60 +/- 30 nM was determined for the dissociation constant. Single photon counting methods were employed to measure the fluorescence lifetime of the fluorescein donor in the presence and absence of HMG1 domain B. These FRET experiments revealed a distance change that was used to estimate a bend angle of 80-95 degrees for the cisplatin-modified DNA upon protein binding. Stopped-flow fluorescence spectroscopic experiments afforded kinetic parameters for HMG1 domain B binding to the cisplatin-modified DNA probe, with kon = 1.1 +/- 0.1 x 10(9) M-1 s-1 and koff = 30 +/- 4 s-1.

RNA-aminoglycoside antibiotic interactions: fluorescence detection of binding and conformational change.

A hammerhead ribozyme has been labeled with a fluorescein reporter dye which enables the nucleic acid to detect binding of small organic compounds such as neomycin. The fluorescent changes are associated with conformational changes in the RNA and can be used to determine the binding modes of the drugs.

Thermodynamics of RNA hairpins containing single internal mismatches.

Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions. Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities. The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site. UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins. Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability. In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities. The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed.

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.

Herpes simplex virus processivity factor UL42 imparts increased DNA-binding specificity to the viral DNA polymerase and decreased dissociation from primer-template without reducing the elongation rate.

Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased approximately 15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA approximately 2-fold faster than did Pol and dissociated approximately 10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, approximately 15 [corrected] nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented.

Mutations that specifically impair the DNA binding activity of the herpes simplex virus protein UL42.

The herpes simplex virus DNA polymerase is a heterodimer consisting of a catalytic subunit and the protein UL42, which functions as a processivity factor. It has been hypothesized that UL42 tethers the catalytic subunit to the DNA template by virtue of DNA binding activity (J. Gottlieb, A. I. Marcy, D. M. Coen, and M. D. Challberg, J. Virol. 64:5976-5987, 1990). Relevant to this hypothesis, we identified two linker insertion mutants of UL42 that were unable to bind to a double-stranded-DNA-cellulose column but retained their ability to bind the catalytic subunit. These mutants were severely impaired in the stimulation of long-chain-DNA synthesis by the catalytic subunit in vitro. In transfected cells, the expressed mutant proteins localized to the nucleus but were nonetheless deficient in complementing the growth of a UL42 null virus. Thus, unlike many other processivity factors, UL42 appears to require an intrinsic DNA binding activity for its function both in vitro and in infected cells. Possible mechanisms for the activity of UL42 and its potential as a drug target are discussed.

A single HMG domain in high-mobility group 1 protein binds to DNAs as small as 20 base pairs containing the major cisplatin adduct.

Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.

HMG domain proteins induce sharp bends in cisplatin-modified DNA.

Circularly permuted linear DNAs of approximately 100 bp were constructed containing the major adduct of the anticancer drug cisplatin, a cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link, at a specific site. Gel electrophoresis mobility shift assays with these probes were used to investigate the effects of binding of HMG domain proteins to the platinated DNAs. The site-specifically platinated duplexes were recognized by six different HMG domain proteins--HMG1, mtTFA, Ixr1, and HMG domains from HMG1 (domain B), mSRY, and LEF-1--with comparable binding affinities (Kd approximately 10(-6) to 10(-7) M). In the presence of the HMG domain proteins, the platinated DNAs were bent significantly more than in their absence, the values being 86 +/- 2 degrees, 87-90 +/- 5 degrees, and 68 +/- 6 degrees, respectively, for the proteins and 65-74 +/- 4 degrees, approximately 50 degrees, and 72 +/- 6 degrees, respectively, for the domains. The variability in bend angles suggests that, although the HMG domain proteins share a common ability to bend platinated DNA, specific contacts between the proteins and the platinated duplex are different. The assay further revealed the bend loci to be centered quite near the platinum adduct. The methodology employed in the present study should be generally applicable for synthesizing other small, circularly permuted, covalently modified DNAs which cannot otherwise be readily obtained.

High-mobility-group 1 protein mediates DNA bending as determined by ring closures.

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.