Toward global availability of low-cost, patient-specific cranial implants: creation and validation of automated CranialRebuild freeware application.

PURPOSE: Financial restrictions limit the options for hermetically precise, patient-specific cranial implants (PSCIs) after decompressive hemicraniectomy (DHC) in low-income countries. Use of image segmentation, modeling software, and 3D printers has lowered costs associated with PSCIs. However, requirements of time and technical expertise have prevented widespread utilization. Our objective was to create a fully automated software algorithm that is able to generate a virtual model (.STL) of a negative of an implant using CT imaging following DHC. METHODS: A freeware algorithm (CranialRebuild) was constructed with the following capabilities: (1) after the upload of digital imaging and communications in medicine files, the normal side is analyzed in reference to the side of DHC, (2) Boolean subtraction is used to obtain a virtual image of the desired implant, and (3) a two-piece virtual model (.STL) of the PSCI mold is generated. In four cadaveric specimens, a standard DHC was performed. Post-DHC CT imaging was used to obtain a .STL of the negative of the implant, which was then printed using poly-lactic acid (PLA). Methylmethacrylate cement was used to generate a PSCI from the mold. The PSCIs were implanted into the index specimens; cosmesis was subjectively evaluated using a 5-point Likert scale. RESULTS: Two specimens were graded as 4/5, indicating that minor post-processing modification was needed for optimal cosmesis. Two specimens were graded as 3/5, indicating that optimal cosmesis could be obtained following moderate post-processing modification. CONCLUSIONS: CranialRebuild can be used to create hermetically precise PSCIs at a fraction of the price of third-party vendors. Validation of this technology has significant implications for the accessibility of customized cranial implants worldwide.

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers.

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

An APP ectodomain mutation outside of the Aß domain promotes Aß production in vitro and deposition in vivo.

Familial Alzheimer's disease (FAD)-linked mutations in the APP gene occur either within the Aß-coding region or immediately proximal and are located in exons 16 and 17, which encode Aß peptides. We have identified an extremely rare, partially penetrant, single nucleotide variant (SNV), rs145081708, in APP that corresponds to a Ser198Pro substitution in exon 5. We now report that in stably transfected cells, expression of APP harboring the S198P mutation (APPS198P) leads to elevated production of Aß peptides by an unconventional mechanism in which the folding and exit of APPS198P from the endoplasmic reticulum is accelerated. More importantly, coexpression of APP S198P and the FAD-linked PS1ΔE9 variant in the brains of male and female transgenic mice leads to elevated steady-state Aß peptide levels and acceleration of Aß deposition compared with age- and gender-matched mice expressing APP and PS1ΔE9. This is the first AD-linked mutation in APP present outside of exons 16 and 17 that enhances Aß production and deposition.

HIV-1 Nef dimers short-circuit immune receptor signaling by activating Tec-family kinases at the host cell membrane.

The HIV-1 virulence factor Nef promotes high-titer viral replication, immune escape, and pathogenicity. Nef interacts with interleukin-2-inducible T-cell kinase (Itk) and Bruton's tyrosine kinase (Btk), two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages, respectively). Using a cell-based bimolecular fluorescence complementation assay, here we demonstrate that Nef recruits both Itk and Btk to the cell membrane and induces constitutive kinase activation in transfected 293T cells. Nef homodimerization-defective mutants retained their interaction with both kinases but failed to induce activation, supporting a role for Nef homodimer formation in the activation mechanism. HIV-1 infection up-regulates endogenous Itk activity in SupT1 T cells and donor-derived peripheral blood mononuclear cells. However, HIV-1 strains expressing Nef variants with mutations in the dimerization interface replicated poorly and were significantly attenuated in Itk activation. We conclude that direct activation of Itk and Btk by Nef at the membrane in HIV-infected cells may override normal immune receptor control of Tec-family kinase activity to enhance the viral life cycle.

Efficacy of Commercial Sanitizers Used in Food Processing Facilities for Inactivation of Listeria Monocytogenes, E. Coli O157:H7, and Salmonella Biofilms.

Bacteria entrapped in biofilms are a source of recurring problems in food processing environments. We recently developed a robust, 7-day biofilm microplate protocol for creating biofilms with strongly adherent strains of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella serovars that could be used to examine the effectiveness of various commercial sanitizers. Listeria monocytogenes 99-38, E.coli O157:H7 F4546, and Salmonella Montevideo FSIS 051 were determined from prior studies to be good biofilm formers and could be recovered and enumerated from biofilms following treatment with trypsin. Extended biofilms were generated by cycles of growth and washing daily, for 7 days, to remove planktonic cells. We examined five different sanitizers (three used at two different concentrations) for efficacy against the three pathogenic biofilms. Quaternary ammonium chloride (QAC) and chlorine-based sanitizers were the least effective, showing partial inhibition of the various biofilms within 2 h (1-2 log reduction). The best performing sanitizer across all three pathogens was a combination of modified QAC, hydrogen peroxide, and diacetin which resulted in ~6-7 log reduction, reaching levels below our limit of detection (LOD) within 1-2.5 min. All treatments were performed in triplicate replication and analyzed by one way repeated measures analysis of variance (RM-ANOVA) to determine significant differences (p < 0.05) in the response to sanitizer treatment over time. Analysis of 7-day biofilms by scanning electron microscopy (SEM) suggests the involvement of extracellular polysaccharides with Salmonella and E. coli, which may make their biofilms more impervious to sanitizers than L. monocytogenes.

Optimization of a Microplate Assay for Generating Listeria Monocytogenes, E. Coli O157:H7, and Salmonella Biofilms and Enzymatic Recovery for Enumeration.

Biofilms enable the persistence of pathogens in food processing environments. Sanitizing agents are needed that are effective against pathogens entrapped in biofilms that are more difficult to inactivate than planktonic cells that are displaced and found on equipment surfaces. We examined conditions to develop, analyze, and enumerate the enhanced biofilms of three different foodborne pathogens assisted by fluorescence adherence assay and enzymatic detachment. We compared three different isomeric forms of fluorescent substrates that are readily taken up by bacterial cells based on carboxy-fluorescein diacetate (5-CFDA, 5,6-CFDA, 5,6-CFDA, SE). Biofilm-forming strains of Escherichia coli O157:H7 F4546 and Salmonella Montevideo FSIS 051 were identified using a microplate fluorescence assay defined previously for L. monocytogenes. Adherence levels were determined by differences in relative fluorescence units (RFU) as well as recovered bacterial cells. Multiple hydrolytic enzymes were examined for each representative pathogen for the most suitable enzyme for detachment and enumeration to confirm adherence data obtained by fluorescence assay. Cultures were grown overnight in microplates, incubated, washed and replenished with fresh sterile growth medium; this cycle was repeated for seven consecutive days to enrich for robust biofilms. Treatments were performed in triplicate and compared by one-way analysis of variance (ANOVA) to determine significant differences (p < 0.05).

Bacterial arginine kinases have a highly skewed distribution within the proteobacteria.

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. A recent search of the available bacterial genomes revealed 49 unique sequences that appear to code for an arginine kinase (AK). The distribution of sequences was highly skewed with thirty nine out the forty nine sequences being found in six Proteobacteria classes (α, ß, δ, γ, ε, and ζ) which represented 46.6% of the 61,335 bacterial genomes available at JGI-IMG/M website. Moreover, twenty one of the unique and metagenome bAK sequences identified were from δ-Proteobacteria despite these representing only 0.88% of the total genomes available. Phylogenetic analyses revealed that the bacterial AK sequences were interpersed between basal species such as cnidarians, sponges and protozoa, displaying an unstable clustering that was dependent upon the parameters chosen for phylogenetic analysis. Three of these putative bacterial AK genes were cloned into the pET45 expression vector, expressed, and biochemically confirmed to be capable of phosphorylating arginine using ATP. Results of the kinetic analyses of the putative bAKs from Ahrensia, D. autotrophicum, and O. profundus show that the catalytic efficiencies with respect to arginine for each enzyme, measured at 104-105 M-1 s-1, fall within the range expected for competent arginine kinases.

Characterization of the arginine kinase isoforms in Caenorhabditis elegans.

Phosphagen kinases (PKs) are well-studied enzymes involved in energy homeostasis in a wide range of animal, protozoan, and even some bacterial species. Recent genome efforts have allowed comparative work on the PKs to extend beyond the biochemistry of individual proteins to the comparative cellular physiology and examining of the role of all PK family members in an organism. The sequencing of the Caenorhabditis elegans genome and availability of sophisticated genetic tools within that system affords the opportunity to conduct a detailed physiological analysis of the PKs from a well known invertebrate for comparison with the extensive work conducted on vertebrate systems. As a first step in this effort we have carried out a detailed molecular genetic and biochemical characterization of the PKs in C. elegans. Our results reveal that C. elegans has five PK genes encoding arginine kinases that range in catalytic efficiency (kcat/KM(Arg)) from (3.1±0.6)×10(4) to (9±4)×10(5) M(-1) s(-1). This range is generally within the range seen for arginine kinases from a variety of species. Our molecular genetic and phylogenetic analysis reveals that the gene family has undergone extensive intron loss and gain within the suborder Rhabditina. In addition, within C. elegans we find evidence of gene duplication and loss. The analysis described here for the C. elegans AKs represents one of the most complete biochemical and molecular genetic analysis of a PK family within a genetically tractable invertebrate system and opens up the possibility of conducting detailed physiological comparisons with vertebrate systems using the sophisticated tools available with this model invertebrate system.