Structural basis for topological regulation of Tn3 resolvase.

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Site-specific DNA recombinases alter the connectivity of DNA by recognizing specific DNA sequences, then cutting the DNA strands and pasting them together in a new configuration. Such enzymes play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful biotechnology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. However, others have evolved elaborate mechanisms to ensure that only one of multiple possible recombination products is produced. Tn3 resolvase has long been known to be regulated by DNA topology­that is, it will cut and reconnect two target sequences only if they lie on the same DNA molecule, and if they are in the proper relative orientation. This study presents new structural and biochemical data that lead to a new, detailed model of the large protein­DNA complex formed by Tn3 resolvase and its cognate sites. This 3D model illustrates how DNA topology can be harnessed to regulate the activity of a recombinase and provides a basis for engineering Tn3 resolvase and related recombination systems as genome editing tools.

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation.

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.

Deciphering the Roles of Multicomponent Recognition Signals by the AAA+ Unfoldase ClpX.

ATP-dependent protein remodeling and unfolding enzymes are key participants in protein metabolism in all cells. How these often-destructive enzymes specifically recognize target protein complexes is poorly understood. Here, we use the well-studied AAA+ unfoldase-substrate pair, Escherichia coli ClpX and MuA transposase, to address how these powerful enzymes recognize target protein complexes. We demonstrate that the final transposition product, which is a DNA-bound tetramer of MuA, is preferentially recognized over the monomeric apo-protein through its multivalent display of ClpX recognition tags. The important peptide tags include one at the C-terminus ("C-tag") that binds the ClpX pore and a second one (enhancement or "E-tag") that binds the ClpX N-terminal domain. We construct a chimeric protein to interrogate subunit-specific contributions of these tags. Efficient remodeling of MuA tetramers requires ClpX to contact a minimum of three tags (one C-tag and two or more E-tags), and that these tags are contributed by different subunits within the tetramer. The individual recognition peptides bind ClpX weakly (KD>70 µM) but impart a high-affinity interaction (KD~1.0 µM) when combined in the MuA tetramer. When the weak C-tag signal is replaced with a stronger recognition tag, the E-tags become unnecessary and ClpX's preference for the complex over MuA monomers is eliminated. Additionally, because the spatial orientation of the tags is predicted to change during the final step of transposition, this recognition strategy suggests how AAA+ unfoldases specifically distinguish the completed "end-stage" form of a particular complex for the ideal biological outcome.

Positron emission tomography with [18F]flutemetamol and [11C]PiB for in vivo detection of cerebral cortical amyloid in normal pressure hydrocephalus patients.

BACKGROUND AND PURPOSE: This study determined the correlation between uptake of the amyloid positron emission tomography (PET) imaging agent [(18) F]flutemetamol and amyloid-ß measured by immunohistochemical and histochemical staining in a frontal cortical biopsy. METHODS: Fifteen patients with possible normal pressure hydrocephalus (NPH) and previous brain biopsy obtained during intracranial pressure monitoring underwent [18F]flutemetamol PET. Seven of these patients also underwent [11C] Pittsburgh compound B (PiB) PET. [18F]Flutemetamol and [11C]PiB uptake was quantified using standardized uptake value ratio (SUVR) with the cerebellar cortex as a reference region. Tissue amyloid-ß was evaluated using the monoclonal antibody 4G8, Thioflavin-S and Bielschowsky silver stain. RESULTS: [18F]Flutemetamol and [11C]PiB SUVRs correlated with biopsy specimen amyloid-ß levels contralateral (r = 0.86, P < 0.0001; r = 0.96, P = 0.0008) and ipsilateral (r = 0.82, P = 0.0002; r = 0.87, P = 0.01) to the biopsy site. Association between cortical composite [(18) F]flutemetamol SUVRs and [11C]PiB SUVRs was highly significant (r = 0.97, P = 0.0003). CONCLUSIONS: [18F]Flutemetamol detects brain amyloid-ß in vivo with moderate to high sensitivity and high specificity. This agent, therefore, represents a valuable new tool to study and verify the presence of amyloid-ß pathology, both in patients with possible NPH and among the wider population.

The µ transpososome structure sheds light on DDE recombinase evolution.

Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of the Mu transpososome--Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA--determined from data that extend anisotropically to 5.2 Å, 5.2 Å and 3.7 Å resolution, in conjunction with previously determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains have different roles when bound to different sites. The structure also provides explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved 'DDE' catalytic domain, comparisons among the limited set of available complex structures indicate that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

Moving DNA around: DNA transposition and retroviral integration.

Mobile DNA elements are found in all kingdoms of life, and they employ numerous mechanisms to move within and between genomes. Here we review recent structural advances in understanding two very different families of DNA transposases and retroviral integrases: the DDE and Y1 groups. Even within the DDE family which shares a conserved catalytic domain, there is great diversity in the architecture of the synaptic complexes formed by the intact enzymes with their cognate element-end DNAs. However, recurring themes arise from comparing these complexes, such as stabilization by an intertwined network of protein-DNA and protein-protein contacts, and catalysis in trans, where each active subunit catalyzes the chemical steps on one DNA segment but also binds specific sequences on the other.

Orchestrating serine resolvases.

A remarkable feature of the serine resolvases is their regulation: the wild-type enzymes will catalyse intra- but not inter-molecular recombination, can sense the relative orientation of their sites and can exchange strands directionally, despite the fact that there is no net release of chemical bond energy. The key to this regulation is that they are only active within a large intertwined complex called the 'synaptosome'. Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a 'topological filter'. Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. The regulatory portion of this complex contains additional copies of the recombinase and sometimes other DNA-bending proteins. We are using a combination of X-ray crystallography, biochemistry and genetics to model the full synaptic complex and to understand how the regulatory portion activates the crossover-site-bound recombinases.

Adrenal gland injury secondary to blunt traumatic mechanisms: a marker of overall injury severity.

INTRODUCTION: Adrenal gland injuries (AGI) are seen increasingly frequently owing to advances in modern imaging techniques. This study describes a series of patients with blunt AGI, with the emphasis on AGI as a marker of injury severity, CT-radiographic classification of AGI and associated injury patterns. MATERIAL AND METHODS: A retrospective review of blunt trauma patients with AGI was performed. Variables examined included demographics, mechanism of injury, length of hospital and ICU stay, clinical status on admission, AGI characteristics, associated injuries, complications, procedures, mortality and discharge disposition. RESULTS: There were 29 AGI patients with a mean injury severity score of 25. The most common injury mechanisms were motor-vehicular collisions (15/29) and falls (5/29). Right-sided AGI (16/29) outnumbered left-sided (12/29) injuries. The most common CT-radiographic types of AGI were adrenal gland contusions and lacerations with limited "blush". While patterns of injuries differed between right and left-sided AGI, the mean number of injuries did not. The most common associated injuries included extremity (21/29), rib (20/29) and spinal fractures (18/29). Common procedures included orthopaedic fixation (10/29), vena cava filter (8/29) and tracheostomy (5/29). A median of two complications per patient was reported, including adrenal insufficiency in two patients. Mortality was 17%. The median hospital and ICU length of stay were 15 and 12 days, respectively. CONCLUSIONS: Adrenal gland injury is associated with significant morbidity and mortality. With modern imaging modalities capable of reliably detecting adrenal injury, the presence of AGI should be considered a marker of overall injury severity. The authors provide a CT-radiographic classification of adrenal injuries.

The dead continue to teach the living: examining the role of computed tomography and magnetic resonance imaging in the setting of postmortem examinations.

High-resolution imaging methods are used more frequently in the setting of postmortem investigation. Used for some time in forensics, computed tomography (CT) and magnetic resonance imaging (MRI) are now being evaluated as complementary or even as alternative means of postmortem examination. We review briefly the history of autopsy and the reasons for the gradual decrease in autopsy rates. An overview of advantages and limitations of modern imaging autopsy techniques is then presented, which includes a discussion of the potential role of imaging autopsy in medical and surgical education. Potential future applications of this technology in postmortem analysis, which includes the incorporation of ultrasound technology, are then discussed.

Cervical spine fractures in geriatric blunt trauma patients with low-energy mechanism: are clinical predictors adequate?

BACKGROUND: Studies have identified clinical predictors to guide radiologic evaluation of the cervical spine in geriatric patients. We hypothesized that clinical predictors are not adequate in the identification of cervical spine fractures in geriatric blunt trauma patients with low-energy mechanism. METHODS: A retrospective case-control study was performed on geriatric blunt trauma patients sustaining low-energy trauma from January 2000 to January 2006. A data form including 8 clinical predictors was completed for each group. RESULTS: There were 35 study and 64 control patients identified. Both groups were similar in age (study 83.6 vs control 81.2) and injury severity score (study 9.06 vs control 9.61). Only neck tenderness exceeded the expected occurrence in the presence of a cervical spine injury (chi(2) = 18.1, P = .001) in just 45.5% of the study group. CONCLUSIONS: Clinical predictors appear inadequate for the evaluation of the cervical spine in geriatric trauma patients with low-energy mechanism.

Application of vacuum-assisted therapy in postoperative ascitic fluid leaks: an integral part of multimodality wound management in cirrhotic patients.

Surgery in patients with hepatic cirrhosis and ascites is associated with significant morbidity, including poor wound healing. Postoperative management of abdominal and perineal wounds in these patients poses a unique challenge owing to increased intra-abdominal pressure, risk for peritonitis, and ascitic fluid leakage. Vacuum-assisted closure (VAC) therapy reportedly improves angiogenesis and epithelialization, controls bacterial contamination, and removes excess tissue fluid. We present 4 cases of successful management of intractable postoperative ascitic fluid leaks utilizing VAC-based techniques. In one case, closure of a profusely draining perineal wound following an abdominoperineal resection was accomplished within 5 days of specialized VAC dressing application. In the other 3 cases, refractory drainage from midline laparotomy incision was successfully managed with the use of VAC therapy. In all 4 cases, the VAC-based system was effective in controlling drainage of ascites and subsequently sealing the wounds. Postoperative use of VAC in conjunction with optimization of medical therapy and judicious tapping of ascites provides a safe and effective method to control ascitic fluid leaks and promote definitive tissue sealing in patients with hepatic cirrhosis.

Complications related to percutaneous endoscopic gastrostomy (PEG) tubes. A comprehensive clinical review.

Percutaneous endoscopic gastrostomy (PEG) has become the modality of choice for providing enteral access to patients who require long-term enteral nutrition. Although generally considered safe, PEG tube placement can be associated with many potential complications. This review describes a variety of PEG tube related complications as well as strategies for complication avoidance. In addition, the reader is presented with a brief discussion of procedures, techniques, alternatives to PEG tubes, and related issues. Special topics covered in this review include PEG tube placement following previous surgery and PEG tube use in pregnancy.

Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus.

In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3' to the CA dinucleotide step, resulting in a reactive 3' OH on one strand and a 5' two base overhang on the complementary strand. In order to investigate the structural properties of the 3' end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3' and 5' flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase.

Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have previously reported that processive DNA synthesis of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is severely compromised by substitution of an Ala for the fingers domain residue Arg 116. In order to further investigate the role of Arg 116 in interactions of MMLV RT with nucleic acids, we have determined the crystal structure of the R116A N-terminal fragment and characterized the binding of two self-complementary DNA duplexes [d(CATGCATG)2 and d(CGCGCGCG)2] to both the wild-type and R116A fragments by isothermal titration calorimetry. The resultant thermodynamic profiles extrapolated to 25 degrees C reveal that binding of the wild-type N-terminal fragment to both DNA duplexes is enthalpy-driven and characterized by an unfavorable entropy. Although the temperature dependence of the respective protein-DNA binding enthalpies is markedly different reflecting distinct heat capacity changes, the binding free energies are nearly identical and relatively invariant to temperature (DeltaG approximately -6.0 kcal x mol(-1)). In contrast to the wild-type fragment, the R116A fragment exhibits no measurable affinity for either DNA duplex, yet its crystal structure reveals no significant changes when compared to the wild-type structures. We suggest that hydrogen-bonding interactions involving the fingers domain residue Arg 116 are critical for DNA binding as well as processive DNA synthesis by MMLV RT.

Characterization of critical interactions between Ndt80 and MSE DNA defining a novel family of Ig-fold transcription factors.

The Ndt80 protein of the yeast Saccharomyces cerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors. The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect beta-strands which form the beta-sandwich fold common to proteins in this superfamily. However, the DNA-binding domain of Ndt80 is considerably larger than many other members of the Ig-fold superfamily and it appears to make a larger number of contacts with the DNA than these proteins. To determine the contribution of each of these contacts and to examine if the mechanism of Ndt80 DNA binding was similar to other members of the Ig-fold superfamily, amino acid substitutions were introduced at each residue that contacts the DNA and assayed for their effect on Ndt80 activity. Many of the mutations caused significant decreases in DNA-binding affinity and transcriptional activation. Several of these are in residues that are not found in other sub-families of Ig-fold proteins. These additional contacts are likely responsible for Ndt80's ability to bind DNA as a monomer while most other members require additional domains or cofactors to recognize their sites.

Sum1 and Ndt80 proteins compete for binding to middle sporulation element sequences that control meiotic gene expression.

A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomyces cerevisiae depends upon induction of the middle sporulation genes. Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence element termed the middle sporulation element (MSE). Sum1 is a transcriptional repressor that binds to MSEs and represses middle sporulation genes during mitosis and early sporulation. We demonstrate that Sum1 and Ndt80 have overlapping yet distinct sequence requirements for binding to and acting at variant MSEs. Whole-genome expression analysis identified a subset of middle sporulation genes that was derepressed in a sum1 mutant. A comparison of the MSEs in the Sum1-repressible promoters and MSEs from other middle sporulation genes revealed that there are distinct classes of MSEs. We show that Sum1 and Ndt80 compete for binding to MSEs and that small changes in the sequence of an MSE can yield large differences in which protein is bound. Our results provide a mechanism for differentially regulating the expression of middle sporulation genes through the competition between the Sum1 repressor and the Ndt80 activator.

Crystallographic studies of a novel DNA-binding domain from the yeast transcriptional activator Ndt80.

The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution.