Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

Response of crypt paneth cells in the small intestine following total-body gamma-irradiation.

Ionizing irradiation causes damage and functional failure of irradiation-sensitive systems and tissues such as small intestine. The molecular mechanisms underlying inflammatory and adaptive responses to acute irradiation damage are poorly understood. Using a mouse model of total-body γ-irradiation, we assessed the irradiation response of crypt host-defense Paneth cells by measuring alpha-defensin 4 (AD4) expression and correlated the gathered data with activation of the caspase-1/IL-1ß inflammatory signaling cascade. The irradiation injury was produced in CD2F1 mice exposed to 9.25 Gy γ-radiation. This dose resulted in 85-100 percent mortality at the 15(th) day post-irradiation. Small intestine tissue samples were collected at the 7th day post-irradiation. Assessment of irradiation-associated pro-inflammatory alterations in small intestine tissue and expression of AD4 in Paneth cells was conducted using confocal immunofluorescence imaging, transmission electron microscopy (TEM), light microscopy, and immunoblotting techniques. The small intestine analysis revealed an increase in the precursor form of IL-1ß, the activated form of IL-1ß, and the activated form of caspase-1 (p10 CASP-1) at the 7(th) day post-irradiation. Immunoprecipitation analysis showed increased interaction between IL-1ß and p10 CASP-1 after irradiation. This effect was observed in the irradiated small intestine and CD15-positive Paneth cells using confocal imaging techniques. The pro-inflammatory alterations in Paneth cells were accompanied by increases in AD4 mRNA and its 8 kD peptide product. Paneth cell secretory activity was observed at the sites of bacterial translocation in the crypt lumens. These data suggest that Paneth cells can contribute to small intestine inflammatory remodeling during the post-irradiation period.

Molecular dynamics simulations of sputtering of Langmuir-Blodgett multilayers by keV C(60) projectiles.

Coarse-grained molecular dynamics computer simulations are applied to investigate fundamental processes induced by an impact of keV C(60) projectile at an organic overlayer composed of long, well-organized linear molecules. The energy transfer pathways, sputtering yields, and the damage induced in the irradiated system, represented by a Langmuir-Blodgett (LB) multilayers composed from molecules of bariated arachidic acid, are investigated as a function of the kinetic energy and impact angle of the projectile and the thickness of the organic system. In particular, the unique challenges of depth profiling through a LB film vs. a more isotropic solid are discussed.The results indicate that the trajectories of projectile fragments and, consequently, the primary energy can be channeled by the geometrical structure of the overlayer. Although, a similar process is known from sputtering of single crystals by atomic projectiles, it has not been anticipated to occur during C(60) bombardment due to the large size of the projectile. An open and ordered molecular structure of LB films is responsible for such behavior. Both the extent of damage and the efficiency of sputtering depend on the kinetic energy, the impact angle, and the layer thickness. The results indicate that the best depth profiling conditions can be achieved with low-energy cluster projectiles irradiating the organic overlayer at large off-normal angles.

Dynamics of molecular impacts on soft materials: from fullerenes to organic nanodrops.

The present theoretical study explores the interaction of various energetic molecular projectiles and clusters with a model polymeric surface, with direct implications for surface analysis by mass spectrometry. The projectile sizes (up to 23 kDa) are intermediate between the polyatomic ions (SF(5), C(60)) used in secondary ion mass spectrometry and the large organic microdroplets generated, for example, in desorption electrospray ionization. The target is a model of amorphous polyethylene, already used in a previous study [Delcorte, A.; Garrison, B. J. J. Phys. Chem. C 2007, 111, 15312]. The chosen method relies on classical molecular dynamics (MD) simulations, using a coarse-grained description of polymeric samples for high energy or long time calculations (20-50 ps) and a full atomistic description for low energy or short time calculations (<1 ps). Two regions of sputtering or desorption are observed depending on the projectile energy per nucleon (i.e., effectively the velocity). The transition, occurring around 1 eV/nucleon, is identified by a change of slope in the curve of the sputtering yield per nucleon vs energy per nucleon. Beyond 1 eV/nucleon, the sputtering yield depends only on the total projectile energy and not on the projectile nuclearity. Below 1 eV/nucleon, i.e., around the sputtering threshold for small projectiles, yields are influenced by both the projectile energy and nuclearity. Deposition of intact molecular clusters is also observed at the lowest energies per nucleon. The transition in the sputtering curve is connected to a change of energy deposition mechanisms, from atomistic and mesoscopic processes to hydrodynamic flow. It also corresponds to a change in terms of fragmentation. Below 1 eV/nucleon, the projectiles are not able to induce bond scissions in the sample. This region of molecular emission with minimal fragmentation offers new analytical perspectives, out of reach of smaller molecular clusters such as fullerenes.

Understanding gold-thiolate cluster emission from self-assembled monolayers upon kiloelectronvolt ion bombardment.

This article focuses on the emission of organometallic clusters upon kiloelectronvolt ion bombardment of self-assembled monolayers. It is particularly relevant for the elucidation of the physical processes underlying secondary ion mass spectrometry (SIMS). The experimental system, an overlayer of octanethiols on gold, was modeled by classical molecular dynamics, using a hydrocarbon potential involving bonding and nonbonding interactions (AIREBO). To validate the model, the calculated mass and energy distributions of sputtered atoms and molecules were compared to experimental data. Our key finding concerns the emission mechanism of large clusters of the form MxAuy up to M6Au5 (where M is the thiolate molecule), which were not observed under sub-kiloelectronvolt projectile bombardment. Statistically, they are predominantly formed in high-yield events, where many atoms, fragments, and (supra)molecular species are desorbed from the surface. From the microscopic viewpoint, these high-yield events mostly stem from the confinement of the projectile and recoil atom energies in a finite microvolume of the sample surface. As a result of the high local energy density, molecular aggregates desorb from an overheated liquidlike region surrounding the impact point of the projectile.

Sputtering of water ice induced by C60 bombardment: onset of plume formation.

The interaction of a 5 keV C(60) projectile with amorphous water ice is studied using molecular dynamics computer simulations. The energetic C(60) molecule causes large-scale collisional events in the subsurface region, involving more than 10(4) water molecules in a time of less than 3 ps. The energy deposited in the sample is sufficiently large to turn the ice into a superheated and superdense gas. The gas is expelled into the vacuum, leading to the formation of a flow that manifests itself in the angular and velocity distributions of emitted water molecules.

Arthroscopic biceps tenodesis using suture anchors through the subclavian portal.

Biceps tenodesis is typically performed through an open anterior incision. Even when an arthroscopic rotator cuff repair is performed, an open procedure is typically performed to address the biceps rupture or subluxation. Recently, there has been great interest in performing this procedure arthroscopically. Techniques have included using an interference screw or 2 suture anchors through an anterior cannula. If the biceps is partially ruptured or subluxated and the proximal end is still visible in the joint, a biceps tenodesis can be performed using standard arthroscopic techniques and suture anchors. The senior author (K.D.N.) developed the subclavian portal in 1997 for arthroscopic repair of rotator cuff tears using a pointed suture grasper. This portal is located 1 to 2 cm medial to the acromioclavicular joint line, directly above and slightly medial to the coracoid. It provides an optimal angle for suture anchor placement directly through the anterior supraspinatus or coracohumeral ligament and into the humeral head at the edge of the articular cartilage. Anchors inserted through the subclavian portal reproduce the 45 degrees Deadman's angle, which was described for placing anchors during rotator cuff repair. Using a burr or shaver through the lateral portal, the articular and bony surface under the biceps tendon and just proximal to the bicipital groove are abraded. Suture anchors are inserted through the subclavian portal, then through the biceps tendon, and into the bone. Sutures are retrieved and tied through the lateral cannula if there is a tear of the supraspinatus. If the supraspinatus is intact, the sutures can be tied intra-articularly through the anterior cannula. Release of the biceps is not performed until the repair is accomplished, which prevents the tendon from retracting down the bicipital groove. The anatomy of the subclavian portal is reviewed and the technique of the arthroscopic biceps tenodesis is presented. Preliminary results of 11 cases with average follow-up of 24 months are presented. Ninety-one percent of the cases had good/excellent results. Adhesive capsulitis occurred in 1 Workers' Compensation patient, which resulted in a fair outcome.

TGF-beta1-enhanced TCP-coated sensate scaffolds can detect bone bonding.

Porous polybutylene terephthalate (PBT) scaffold systems were tested as orthopedic implants to determine whether these scaffolds could be used to detect strain transfer following bone growth into the scaffold. Three types of scaffold systems were tested: porous PBT scaffolds, porous PBT scaffolds with a thin beta-tricalcium phosphate coating (LC-PBT), and porous PBT scaffolds with the TCP coating vacuum packed into the scaffold pores (VI-PBT). In addition, the effect of applying TGF-beta1 to scaffolds as an enhancement was examined. The scaffolds were placed onto the femora of rats and left in vivo for 4 months. The amount of bone ingrowth and the strain transfer through various scaffolds was evaluated by using scanning electron microscopy, histology, histomorphometry, and cantilever bend testing. The VI-PBT scaffold showed the highest and most consistent degree of mechanical interaction between bone and scaffold, providing strain transfers of 68.5% (+/-20.6) and 79.2% (+/-8.7) of control scaffolds in tension and compression, respectively. The strain transfer through the VI-PBT scaffold decreased to 29.1% (+/-24.3) and 30.4% (+/-25.8) in tension and compression when used with TGF-beta1. TGF-beta1 enhancement increased the strain transfer through LC-PBT scaffolds in compression from 9.4% (+/-8.7) to 49.7% (+/-31.0). The significant changes in mechanical strain transfer through LC-PBT and VI-PBT scaffolds correlated with changes in bone ingrowth fraction, which was increased by 39.6% in LC-PBT scaffolds and was decreased 21.3% in VI-PBT scaffolds after TGF-beta1 enhancement. Overall, the results indicate that strain transfer through TCP-coated PBT scaffolds correlate with bone ingrowth after implantation, making these instrumented scaffolds useful for monitoring bone growth by monitoring strain transfer.

Molecular dynamics simulation of the laser disintegration of aerosol particles.

The mechanisms of disintegration of submicrometer particles irradiated by short laser pulses are studied by a molecular dynamics simulation technique. Simulations at different laser fluences are performed for particles with homogeneous composition and particles with transparent inclusions. Spatially nonuniform deposition of laser energy is found to play a major role in defining the character and the extent of disintegration. The processes that contribute to the disintegration include overheating and explosive decomposition of the illuminated side of the particle, spallation of the backside of large particles, and disruption of the transparent inclusion caused by the relaxation of the laser-induced pressure. The observed mechanisms are related to the nature of the disintegration products and implications of the simulation results for aerosol time-of-flight mass spectrometry are discussed. Application of multiple laser pulses is predicted to be advantageous for efficient mass spectrometry sampling of aerosols with a large size to laser penetration depth ratio.

Molecule liftoff from surfaces.

Molecular dynamics simulations have been used to model the kiloelectronvolt particle bombardment of organic layers on metal substrates such as occurs in the analytical techniques of secondary ion mass spectrometry and fast atom bombardment mass spectrometry. Vignettes of insights gained from the simulations along with comparisons to experimental data are presented in this Account. Topics include intact molecular ejection vs fragmentation, prediction of reaction pathways, influence of the substrate, and quantitative predictions of energy and angular distributions.

Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways.

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5(ts). Biochemical analysis of API import in the apg5(ts) strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in the apg5(ts) mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.

Evaluation of a rapid slide agglutination test for identification of Staphylococcus aureus.

This study compared the Staph Latex Kit (Remel) with two other rapid agglutination tests (Staphaurex Plus (Murex) and Staphyloslide (Becton Dickinson Microbiological Systems)) and the tube coagulase test. The Staph Latex Kit, Staphaurex Plus, Staphyloslide, and Tube Coagulase correctly identified 98.4%, 100%, 99.5%, and 99.5%, of 191 staphylococcal isolates, respectively.

Consideration of the pH-dependent inhibition of dihydrofolate reductase by methotrexate.

Poisson-Boltzmann calculations were used to determine the pKa of protein functional groups in the unliganded dihydrofolate reductase enzyme, and the pKa of protein and ligand groups in methotrexate-enzyme complexes. The results reported here are in conflict with two fundamental tenets of dihydrofolate reductase inhibition by methotrexate: (1) Asp27 is not expected to be protonated near pH 6.5 in the apoenzyme as previously proposed based on fitting of empirical equations to binding data, and (2) the calculated pKa for the pteridine N1 of the inhibitor while bound to the protein is significantly lower than that estimated for this group from interpretation of NMR data (>10). In fact, the electrostatic calculations and complementary quantum chemical calculations indicate that Asp27 is likely protonated when methotrexate is bound, resulting in a neutral dipole-dipole interaction rather than a salt-bridge between the enzyme and the inhibitor. Reasons for this discrepancy with the experimental data are discussed. Furthermore, His45 and Glu17 in the Escherichia coli enzyme are proposed to be in part responsible for the pH dependence of the conformational degeneracy in the inhibitor-enzyme complex.

Reconstitution in vitro of the V1 complex from the yeast vacuolar proton-translocating ATPase. Assembly recapitulates mechanism.

Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures. Assembly of soluble V1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits. Specifically, V1 can be assembled directly from complex III (subunits E and G) with complex IV (subunits A, B, D, and F) without prior disassembly of complex IV. The formation of complex III in vivo is also shown to be essential and could not be achieved in vitro. Assembly from simpler precursors is possible and is enhanced by added ATP. Assembly can be blocked by N-ethylmaleimide in a Vma1p (subunit A)-specific manner. From these data, we extend our previous model to consider an assembly pathway whose steps reflect the catalytic mechanism of the Boyer binding-change model.

Medical necessity of orthognathic surgery for the treatment of dentofacial deformities associated with temporomandibular disorders.

This retrospective study assessed the medical necessity of orthognathic surgery for the treatment of dentofacial deformities associated with temporomandibular joint disorders from a perspective of cost effectiveness, myofascial and masticatory function, and quality of life. Questionnaires were mailed to 385 orthognathic surgical patients from one oral and maxillofacial surgery practice. Each questionnaire consisted of three sections: medical costs associated with the patient's orofacial symptoms, symptoms associated with the patient's condition, and the patient's quality of life. One hundred fifty patients responded to the questionnaire. On average, patients spent less money per month on costs associated with their orofacial problem after surgery, and there were fewer visits to the doctor postoperatively. All temporomandibular symptoms were significantly reduced postoperatively, with the exception of facial pain. Quality of life also showed improvement postoperatively. Although orthognathic surgery may be of slightly greater benefit to specific groups of patients based on occlusal relationship and age, all patients in this study showed significant postoperative improvement.

Symptomatic Shigella sonnei urinary tract infection.

The clinical course for a patient with symptomatic urinary tract infection due to Shigella sonnei is described. The role of Shigella spp. as urinary pathogens is reviewed.

Average blood loss and the risk of requiring perioperative blood transfusion in 506 orthognathic surgical procedures.

PURPOSE: This study quantifies the estimated blood loss in seven groups of orthognathic surgical procedures and the risk of requiring perioperative blood transfusion, and identifies the factors relating to blood loss and need for transfusion. PATIENTS AND METHODS: The records of 506 consecutive patients who underwent various orthognathic surgical procedures under hypotensive anesthesia from 1987 to 1990 were analyzed retrospectively. All procedures were performed by the authors in the same hospital setting. Patients were placed in seven groups based on the operation(s) performed, including single-jaw and double-jaw procedures. Estimated blood loss was calculated for each group and was correlated with patients' sex, age, and year of surgery, and whether they autodonated blood. The volume of intravenous (IV) fluids given was also recorded. RESULTS: Average estimated blood loss for all groups was 273.23 mL. Double-jaw procedures resulted in more blood loss than single-jaw procedures. Men and boys had a higher average blood loss than women and girls, but average blood loss was not affected significantly by patients' age or year of surgery. Only four patients (0.8%) received blood transfusions, and their average estimated blood loss was 975 mL. The percentage of patients autodonating increased from 10.4% in 1987 to 54.9% in 1990. There was no significant difference in the percentage of autodonators by surgical procedure. CONCLUSIONS: The need for blood transfusion in this study was extremely low. Factors contributing to this are believed to be use of hypotensive anesthesia; a single surgical team; and a constant surgical setting. Patients having double-jaw surgery are at greater risk for blood loss than those having single-jaw procedures, and should be so advised, along with the risks of blood transfusion. The authors believe that under the conditions of this study the use of autodonation is not necessary, with the possible exception of complex double-jaw procedures involving small patients.

Potential energy surfaces for chemical reactions at solid surfaces.

Many-body potential energy surfaces (PESs) for describing atomic interactions in gas-solid and surface reaction dynamics are reviewed in this work. Initial PESs from the 1960s-1970s were restricted to a diatomic molecule interacting with a solid surface. Since the 1980s, a multitude of many-body reactive PESs, their parameterization, and their applications have been reported in the literature. Although we mention most of the PESs in general, we have chosen to describe only those that either have had general utility or have had staying power, i.e. they have been used widely by other research groups. The potentials discussed in the most detail are the Stillinger-Weber and Tersoff Si PESs, the Brenner hydrocarbon PES, and the embedded-atom method (EAM) style potentials for metals. We conclude that although these PESs have been used successfully in large-scale computer simulations, further development is needed in many-body PESs. In particular, the development of new functional forms for multicomponent reactive systems is required.

Utilization of 3-D computer tomographs in oral surgery.

Imaging is essential in the diagnosis and treatment of maxillofacial abnormalities. CT scans are but one type of imaging studies available to the practitioner. Computers have recently enhanced the traditional CT scan so that it can now be viewed in a three-dimensional reconstruction. Essentials of 3-D imaging and its uses will be presented by the authors, with various case reports to illustrate their value in the dental office.

Molecular dynamics simulations of dimer opening on a diamond {001}(2x1) surface.

Computer simulations of hydrocarbon and related molecules using empirical force fields have become important tools for studying a number of biological and related processes at the atomic scale. Traditional force fields, however, cannot be used to simulate dynamic chemical reactivity that involves changes in atomic hybridization. Application of a many-body potential function allows such reactivity to occur in a computer simulation. Simulations of the reaction of small hydrocarbon molecules adsorbed on a reconstructed diamond {001}(2x1) surface suggest that these hydrocarbons are highly reactive species and that initial stages of diamond growth proceed through a dimer-opening mechanism. Rates estimated from transition state theory of two interconversions between states where the dimer is open and closed are given.