Posterior mesh inguinal hernia repairs: a propensity score matched analysis of laparoscopic and robotic versus open approaches.

PURPOSE: International guidelines suggest the use of lapro-endoscopic technique for primary unilateral inguinal hernia (IHR) because of lower postoperative pain and reduction in chronic pain. It is unclear if the primary benefit is due to the minimally invasive approach, the posterior mesh position or both. Further research evaluating posterior mesh placement using open preperitoneal techniques is recommended. A potential benefit of open preperitoneal repair is the avoidance of general anesthesia, as these repairs can be performed under local anesthesia. This study compares clinical and patient-reported outcomes after unilateral laparo-endoscopic, robotic, and open posterior mesh IHRs. METHODS: We performed a propensity score matched analysis of patients undergoing IHR between 2012 and 2021 in the Abdominal Core Health Quality Collaborative registry. 10,409 patients underwent a unilateral IHR via a posterior approach. Hernia repairs were performed via minimally invasive surgery (MIS) which includes laparoscopic and robotic transabdominal preperitoneal (TAPP), laparoscopic totally extraperitoneal (TEP), or open transrectus preperitoneal/open preperitoneal (TREPP/OPP) approaches. Propensity score matching (PSM) utilizing nearest neighbor matching accounted for differences in baseline characteristics and possible confounding variables between groups. We matched 816 patients in the MIS cohort with 816 patients in the TREPP/OPP group. Outcomes included patient reported quality of life, hernia recurrence, and postoperative opioid use. RESULTS: Improvement was seen after TREPP/OPP as compared to MIS IHR in EuraHS at 30 days (Median(IQR) 7.0 (2.0-16.64) vs 10 (2.0-24.0); OR 0.69 [0.55-0.85]; p = 0.001) and 6 months (1.0 (0.0-4.0) vs 2.0 (0.0-4.0); OR 0.63 [0.46-85]; p = 0.002), patient-reported opioid use at 30-day follow-up (18% vs 45% OR 0.26 [0.19-0.35]; p < 0.001), and rates of surgical site occurrences (0.8% vs 4.9% OR 0.16 [0.06-0.35]; p < 0.001). There were no differences in EuraHS scores and recurrences at 1 year. CONCLUSIONS: This study demonstrates a potential benefit of open posterior mesh placement over MIS repair in short-term quality of life and seroma formation with equivalent rates of hernia recurrence. Further study is needed to better understand these differences and determine the reproducibility of these findings outside of high-volume specialty centers.

Are perioperative outcomes in cancer-related ventral incisional hernia repair worse than in the general population? An Abdominal Core Health Quality Collaborative (ACHQC) database study.

PURPOSE: Patients with a history of cancer-related abdominal surgery undergoing incisional hernia repair (IHR) are highly heterogenous and increasingly prevalent. We explored whether cancer surgery should be considered an independent risk factor for worse IHR perioperative outcomes. METHODS: Patients undergoing IHR between 2018 and 2020 were identified within the Abdominal Core Health Quality Collaborative (ACHQC). Regression models were used to assess associations between cancer operation history and 30 d surgical site occurrences-exclusive of infection (SSO-EIs), surgical site infections (SSIs), reoperations, time to recurrence, and quality of life (QoL) scores. Cancer cohort subgroup analysis was performed for operative approach and mesh location. RESULTS: 8019 patients who underwent IHR were identified in the ACHQC, 1321 of which had a history of cancer operation. Cancer cohort patients were more likely to be older, males with a higher ASA status and lower BMI, and have longer and wider hernias (p < 0.001). After adjusting for confounding, the cancer cohort was less likely to experience SSO-EIs (OR 0.74, 95% CI 0.59-0.94 p = 0.0092) and showed lower odds of SSIs, reoperations, and recurrence (SSI OR 0.7, 95% CI 0.47-1.05, p = 0.0542; reoperation OR 0.66, 95% CI 0.37-1.17, p = 0.1002; recurrence OR 0.8, 95% CI 0.63-1.02, p = 0.08). There was no difference in postoperative QoL scores between cohorts. There were also no differences in perioperative or QoL outcomes within the cancer cohort based on operative approach or mesh location. CONCLUSION: These data show no evidence that history of cancer operation predisposes patients to worse incisional hernia repair perioperative or quality of life outcomes.

Surgical management of infected abdominal wall mesh: an analysis using the American Hernia Society Quality Collaborative.

INTRODUCTION: Several management strategies exist for the treatment of infected abdominal mesh. Using the American Hernia Society Quality Collaborative, we examined management patterns and 30-day outcomes of infected mesh removal with concomitant incisional hernia repair. METHODS: All patients undergoing incisional hernia repair with removal of infected mesh were identified. A complete repair (CR) was defined as fascial closure with mesh; a partial repair (PR) was defined as fascial closure without mesh or no fascial closure with mesh. A two-tailed p value less than or equal to 0.05 was considered statistically significant. RESULTS: A total of 282 patients were identified: 136 patients in CR group and 146 patients in PR group. Patients had similar comorbidities but differed in wound class (class IV: 55% CR vs 83% SR, p < 0.001) and incidence of associated concomitant colorectal procedures (5% CR vs 18% SR, p = 0.015). Sublay placement was used primarily in CR (94%) compared to PR (52% inlay, 48% sublay). When comparing CR to PR, length of stay (median 6, p = 0.69), complications (40% vs 44%, p = 0.44), surgical site infections (16% vs 21%, p = 0.27), surgical site occurrence (30% vs 35%, p = 0.45), and readmission within 30 days (9% vs. 13%) were not statistically different. CONCLUSIONS: Analysis of data from a multicenter hernia registry comparing CR and PR during infected mesh removal and concurrent incisional hernia repair has not identified higher rates of short-term complications between groups in the presence of infection.

Is there an advantage to laparoscopy over open repair of primary umbilical hernias in obese patients? An analysis of the Americas Hernia Society Quality Collaborative (AHSQC).

PURPOSE: The most common techniques used to repair umbilical hernias are open and laparoscopic. As the obesity epidemic in the United States is growing, it is essential to understand how this morbidity affects umbilical hernia repairs. This study compares laparoscopic versus open umbilical hernia repairs in obese patients. METHODS: All patients with body mass index (BMI) ≥ 30 kg/m2 who underwent elective, open or laparoscopic repair of a primary umbilical hernia with mesh were identified from the Americas Hernia Society Quality Collaborative (AHSQC). A retrospective review of the prospectively collected data was conducted. Outcomes of interest included surgical site infections (SSI), surgical site occurrences requiring procedural intervention (SSOPI), hernia-related quality-of-life survey (HerQles), and long-term recurrence. A logistic regression model was used to generate propensity scores. RESULTS: Of 1507 patients who met the inclusion criteria, 322 were laparoscopic, and 1185 were open cases. The laparoscopic group had higher mean BMI (37 ± 6 vs. 35 ± 5 kg/m2 , P < 0.001 ) and mean hernia width (3 cm ± 1 vs. 2 cm ± 2, P < 0.001). Using a propensity score model, we controlled for several clinically relevant covariates. Propensity score adjustment showed no differences in the 30-day HerQles score (OR 0.93, 95% CI 0.58-1.49), SSI (OR 1.57, 95% CI 0.52-4.77), SSOPI (OR 2.85, 95% CI 0.84-9.62) or hernia recurrence (hazard ratio 0.86, 95% CI 0.50-1.49). CONCLUSION: In obese patients with primary umbilical hernias, there is likely no benefit to laparoscopy over open umbilical hernia repair with mesh with regard to wound morbidity. Although, the long-term recurrence also showed no difference between these two approaches, overall follow up was lacking.

Intraperitoneal versus retromuscular mesh placement for open incisional hernias: an analysis of the Americas Hernia Society Quality Collaborative.

BACKGROUND: Incisional hernia repair with mesh improves long-term outcomes, but the ideal mesh position remains unclear. This study compared intraperitoneal versus retromuscular or preperitoneal sublay (RPS) mesh positions for open incisional hernia repairs. METHODS: All patients who had elective open incisional hernia repairs were identified retrospectively in the Americas Hernia Society Quality Collaborative database. The primary outcome was the rate of 30-day surgical-site infection (SSI). Other outcomes of interest included 30-day surgical-site occurrences requiring procedural intervention (SSOPI), hernia-related quality-of-life survey (HerQLes) scores and long-term recurrence. A logistic model was used to generate propensity scores for mesh position using several clinically relevant co-variables. Regression models adjusting for propensity score and baseline characteristics were developed to assess the effect of mesh placement. RESULTS: A total of 4211 patients were included in the study population: 587 had intraperitoneal mesh and 3624 had RPS mesh. Analysis with propensity score adjustment provided no evidence for differences in SSOPI (odds ratio (OR) 0·79, 95 per cent c.i. 0·49 to 1·26) and SSI (OR 0·91, 0·50 to 1·67) rates or HerQLes scores at 30 days (OR 1·20, 0·79 to 1·82), or recurrence rates (hazard ratio 1·28, 0·90 to 1·82). CONCLUSION: Mesh position had no effect on short- or long-term outcomes, including SSOPI and SSI rates, HerQLes scores and long-term recurrence rates.

ANTECEDENTES: La reparación de una eventración con malla mejora los resultados a largo plazo, pero sigue sin estar definida cuál es la posición ideal de colocación de la malla. Este estudio comparó los resultados de la reparación abierta de una eventración con malla en posición intraperitoneal versus retromuscular o preperitoneal (retromuscular or preperitoneal sublay, RPS). MÉTODOS: Se identificaron de forma retrospectiva todos los pacientes a los que se reparó una eventración por via abierta en el Americas Hernia Society Quality Collaborative. La variable principal fue la tasa de infección de la herida quirúrgica (surgical site infections, SSI) a los 30 días. Se analizaron también las incidencias acaecidas en la herida que hubieran precisado algún tratamiento (surgical site occurrences requiring procedural intervention, SSOPI) dentro de los 30 días postintervención, los resultados de una encuesta de calidad de vida relacionada con la hernia (HerQles) y la recidiva a largo plazo. Se utilizó un modelo logístico con diferentes covariables clínicas relevantes para generar puntajes de propensión con respecto a la posición de malla. Para analizar el efecto de la posición de la malla, se desarrollaron diferentes modelos de regresión ajustados por las características basales y el puntaje de propensión. RESULTADOS: Se incluyeron en el estudio 4.211 pacientes, 587 con malla intraperitoneal y 3.624 con malla RPS. El análisis con ajuste por puntaje de propensión no mostró diferencias en SSOPI (razón de oportunidades, odds ratio, OR 0,624, i.c. del 95% 0,364-1,07), SSI (OR 0,927, i.c. del 95% 0,475-1,81), puntuación HerQles a 30 días (OR 1,19, i.c. del 95% 0,79-1,8) o en el índice de recidivas (OR 1,28, i.c. del 95% 0,897-1,82). CONCLUSIÓN: La posición de la colocación de la malla no tuvo efecto en los resultados a corto o largo plazo, incluidas las tasas de SSOPI y SSI, las puntuaciones de HerQles y la tasa de recidiva a largo plazo.

Hernia recurrence inventory: inguinal hernia recurrence can be accurately assessed using patient-reported outcomes.

PURPOSE: Relying solely on in-person encounters to assess long-term outcomes of hernia repair leads to substantial loss of information and patients lost-to-follow-up, hindering research and quality improvement initiatives. We aimed to determine if inguinal hernia recurrences could be assessed using the Ventral Hernia Recurrence Inventory (VHRI), a previously existing patient-reported outcome (PRO) tool that can be administered through the telephone and has already been validated for diagnosing ventral hernia recurrence. METHODS: A prospective, multicentric comparative study was conducted. Adult patients from two centers (United States and Brazil) at least 1 year after open or minimally invasive inguinal hernia repair were asked to answer the questions of the VHRI in relation to their prior repair. A physical exam was then performed by a blinded surgeon. Testing characteristics and diagnostic performance of the PRO were calculated. Patients with suspected recurrences were preferentially recruited. RESULTS: 128 patients were enrolled after 175 repairs. All patients answered the VHRI and were further examined, where a recurrence was present in 32% of the repairs. Self-reported bulge and patient perception of a recurrence were highly sensitive (84-94%) and specific (93-94%) for the diagnosis of an inguinal hernia recurrence. Test performance was similar in the American and Brazilian populations despite several baseline differences in demographic and clinical characteristics. CONCLUSION: The VHRI can be used to assess long-term inguinal hernia recurrence and should be reestablished as the Hernia Recurrence Inventory (HRI). Its implementation in registries, quality improvement efforts, and research could contribute to improving long-term follow-up rates in hernia patients.

Reversible morphological changes of assembled supramolecular amphiphiles triggered by pH-modulated host-guest interactions.

Reversible template-directed micellar-size and shape modulation by virtue of host-guest reversible docking of molecular templates at the micellar-solvent interface was achieved in water. By combining a π-electron deficient bipyridinium-based gemini amphiphile which is capable of binding and aligning with a π-electron rich tri(ethylene glycol)-disubstituted 1,5-diaminonaphthalene, a switchable detergent system which operates through the pH-responsive formation of bisammonium dications was realised. The binding of the 1,5-diaminonaphthalene guest to the bipyridinium-based micellar aggregate superstructure can be actuated by the addition of acid and base. Upon the addition of acid, protonation of the guest forming the dication deactivates molecular recognition with the charged head groups of the micellar aggregate by Coulombic repulsion. This process is completely reversible upon the addition of base, whereby the guest reintercalates into the superstructure -again forming donor-acceptor π-π stacks at the micellar-solvent interface amongst contiguous surfactant head groups. Synchrotron small angle X-ray scattering and dynamic laser light scattering confirm that this form of reversible directionally-templated micellisation results in an oblate spheroid-to-lamellar micelle morphological transition with a stabilising net decrease in the free energy of micellisation of 1.4 kcal mol(-1) per hydrophobic tail.

Integration of programmable microfluidics and on-chip fluorescence detection for biosensing applications.

We describe the integration of an actively controlled programmable microfluidic sample processor with on-chip optical fluorescence detection to create a single, hybrid sensor system. An array of lifting gate microvalves (automaton) is fabricated with soft lithography, which is reconfigurably joined to a liquid-core, anti-resonant reflecting optical waveguide (ARROW) silicon chip fabricated with conventional microfabrication. In the automaton, various sample handling steps such as mixing, transporting, splitting, isolating, and storing are achieved rapidly and precisely to detect viral nucleic acid targets, while the optofluidic chip provides single particle detection sensitivity using integrated optics. Specifically, an assay for detection of viral nucleic acid targets is implemented. Labeled target nucleic acids are first captured and isolated on magnetic microbeads in the automaton, followed by optical detection of single beads on the ARROW chip. The combination of automated microfluidic sample preparation and highly sensitive optical detection opens possibilities for portable instruments for point-of-use analysis of minute, low concentration biological samples.

A neutral redox-switchable [2]rotaxane.

A limited range of redox-active, rotaxane-based, molecular switches exist, despite numerous potential applications for them as components of nanoscale devices. We have designed and synthesised a neutral, redox-active [2]rotaxane, which incorporates an electron-deficient pyromellitic diimide (PmI)-containing ring encircling two electron-rich recognition sites in the form of dioxynaphthalene (DNP) and tetrathiafulvalene (TTF) units positioned along the rod section of its dumbbell component. Molecular modeling using MacroModel guided the design of the mechanically interlocked molecular switch. The binding affinities in CH(2)Cl(2) at 298 K between the free ring and two electron-rich guests--one (K(a) = 5.8 × 10(2) M(-1)) containing a DNP unit and the other (K(a) = 6.3 × 10(3) M(-1)) containing a TTF unit--are strong: the one order of magnitude difference in their affinities favouring the TTF unit suggested to us the feasibility of integrating these three building blocks into a bistable [2]rotaxane switch. The [2]rotaxane was obtained in 34% yield by relying on neutral donor-acceptor templation and a double copper-catalysed azide-alkyne cycloaddition (CuAAC). Cyclic voltammetry (CV) and spectroelectrochemistry (SEC) were employed to stimulate and observe switching by this neutral bistable rotaxane in solution at 298 K, while (1)H NMR spectroscopy was enlisted to investigate switching upon chemical oxidation. The neutral [2]rotaxane is a chemically robust and functional switch with potential for applications in device settings.

Effects of carbon dioxide, an octenol/phenol mixture, and their combination on tabanidae (Diptera) collections from French 2-tier box traps.

The French 2-tier box trap was evaluated in northwestern Florida to determine its attractiveness to host-seeking tabanids (Diptera) when baited with either CO2 alone; a 4:1:8 mixture of octenol, 3-n-propylphenol, and 4-methylphenol; or a combination of CO2 plus 4:1:8 mixture. Six genera representing 43 species were obtained from this trap. Chrysops and Tabanus made up 10.3 and 87.4% of the total, respectively, with Chrysops celatus Pechuman and Tabanus pallidescens Philip as the predominate species from these two genera. CO2 alone and in combination with the octenol/phenol mixture enhanced overall tabanid collections three- to four-fold compared with no odor. Generally, more species were collected in the top tier of the French 2-tier trap regardless of treatment. Those species that showed a significant difference between top and bottom tiers in traps without odors continued this trend when traps were baited with the semiochemicals. However, Tabanus mixis Philip showed no tier preference in traps baited by CO2 alone, whereas Tabanus lineola F. showed no stratification difference with or without odors.

Electrostatic effects on the free-energy balance in folding a ribosome-inactivating protein.

Electrostatics of globular proteins provides structural integrity as well as specificity of biological function. This dual role is particularly striking for ricin A-chain (RTA), an N-glycosidase which hydrolyzes a single adenine base from a conserved region of rRNA. The reported X-ray crystallographic structure of the RTA mutant E177A demonstrated a remarkable rescue of charge balance in the active site, achieved by the rotation of a second glutamic acid (Glu-208) into the vacated space. To understand this conformational reorganization, molecular-dynamics simulations were applied to estimate relative free energies that govern the thermodynamic stability of E177A together with mutants E177Q and E177D. The simulations anticipate that while E177A is a non-conservative substitution, the protein is more stable than the other two mutants. However, the structural plasticity of the RTA active site is not obtained penalty-free, rather E177A among the mutants shows the largest unfavorable net change in the electrostatic contribution to folding. Of the E177A folded state, reorganization of Glu-208 lowers the electrostatic cost of the free-energy change, yet interestingly, protein interactions oppose the rotational shift, while solvent effects favor the transition.

Implicit attitude formation through classical conditioning.

We sought to demonstrate that attitudes can develop through implicit covariation detection in a new classical conditioning paradigm. In two experiments purportedly about surveillance and vigilance, participants viewed several hundred randomly presented words and images interspersed with critical pairings of valenced unconditioned stimuli (USs) with novel conditioned stimuli (CSs). Attitudes toward the novel objects were influenced by the paired USs: In a surprise evaluation task, the CS paired with positive items was evaluated more positively than the CS paired with negative items. This attitudinal conditioning effect was found using both an explicit measure (Experiments 1 and 2) and an implicit measure (Experiment 2). In a covariation estimation task involving the stimuli presented in the conditioning procedure, participants displayed no explicit memory for the pairings.

Calculations of free-energy contributions to protein-RNA complex stabilization.

The problem of calculating binding affinities of protein-RNA complexes is addressed by analyzing a computational strategy of modeling electrostatic free energies based on a nonlinear Poisson-Boltzmann (NLPB) model and linear response approximation (LRA). The underlying idea is to treat binding as a two-step process. Solutions to the NLPB equation calculate free energies arising from electronic polarizability and the LRA is constructed from molecular dynamics simulations to model reorganization free energies due to conformational transitions. By implementing a consistency condition of requiring the NLPB model to reproduce the solute-solvent free-energy transitions determined by the LRA, a "macromolecule dielectric constant" (epsilon(m)) for treating reorganization is obtained. The applicability of this hybrid approach was evaluated by calculating the absolute free energy of binding and free-energy changes for amino acid substitutions in the complex between the U1A spliceosomal protein and its cognate RNA hairpin. Depending on the residue substitution, epsilon(m) varied from 3 to 18, and reflected dipolar reorientation not included in the polarization modeled by epsilon(m) = 2. Although the changes in binding affinities from substitutions modeled strictly at the implicit level by the NLPB equation with epsilon(m) = 4 reproduced the experimental values with good overall agreement, substitutions problematic to this simple treatment showed significant improvement when solved by the NLPB-LRA approach.

In vivo 4-androstene-3,17-dione and 4-androstene-3 beta,17 beta-diol supplementation in young men.

To determine if known androgenic hormone precursors for testosterone in the androgen pathway would be readily transformed to testosterone, eight male subjects [mean age 23.8 (SEM 3) years, bodymass 83.1 (SEM 8.7) kg, height 175.6 (SEM 8.5) cm] underwent a randomized, double-blind, cross-over, placebo-controlled oral treatment with 200 mg of 4-androstene-3,17-dione (delta 4), 4-androstene-3 beta,17 beta-diol (delta 4 Diol), and placebo (PL). The periods of study were separated by 7 days of washout. Blood was drawn at baseline and subsequently every 30 min for 90 min after treatment. Analysis revealed mean area-under-the-curve (AUC) serum delta 4 concentrations to be higher during delta 4 treatment [2177 (SEM 100) nmol.l-1] than delta 4Diol [900 (SEM 96) nmol.l-1] or PL [484 (SEM 82) nmol.l-1; P < 0.0001]. The delta 4 treatment also revealed a significant effect on total testosterone with a mean AUC [1632.5 (SEM 121) nmol.l-1] that was greater than PL [1418.5 (SEM 131) nmol.l-1; P < 0.05] but not significantly different from those observed after delta 4Diol treatment [1602.9 (SEM 119) nmol.l-1; P = 0.77]. Free testosterone concentrations followed a similar pattern where mean AUC for the delta 4 treatment [6114.0 (SEM 600) pmol.l-1] was greater than after PL [4974.6 (SEM 565) pmol.l-1; P < 0.06] but not significantly different from those observed after delta 4Diol [5632.0 (SEM 389) pmol.l-1; P = 0.48]. The appearance and apparent conversion to total and free testosterone over 90 min was stronger for the delta 4 treatment (r = 0.91, P < 0.045) than for delta 4Diol treatment (r = 0.69, NS) and negatively correlated for PL (r = -0.90, P < 0.02). These results would suggest that delta 4, and perhaps delta 4Diol, taken by month are capable of producing in vivo increases in testosterone concentrations in apparently healthy young men as has already been observed in women after treatment with delta 4.

Modeling implicit reorganization in continuum descriptions of protein-protein interactions.

The determination of free energies that govern protein-protein recognition is essential for a detailed molecular understanding of biological specificity. Continuum models of macromolecular interactions, in which the solvent is treated by an implicit representation and the proteins are treated semi-microscopically, are computationally tractable for estimating free energies, yet many questions remain concerning their accuracy. This article reports a continuum analysis of the free-energy changes underlying the binding of 31 interfacial alanine substitutions of two complexes of the antihen egg white lysozyme (HEL) antibody D1.3 bound with HEL or the antibody E5.2. Two implicit schemes for modeling the effects of protein and solvent relaxation were examined, in which the protein environment was treated as either homogeneous with a "protein dielectric constant" of epsilon(p) = 4 or inhomogeneous, with epsilon(p) = 4 for neutral residues and epsilon(p) = 25 for ionized residues. The results showed that the nonuniform dielectric model reproduced the experimental differences better, with an average absolute error of +/-1.1 kcal/mol, compared with +/-1.4 kcal/mol for the uniform model. More importantly, the error for charged residues in the nonuniform model is +/-0.8 kcal/mol and is nearly half of that corresponding to the uniform model. Several substitutions were clearly problematic in determining qualitative trends and probably required explicit structural reorganization at the protein-protein interface.

Seasonal distribution and abundance of ticks (Acari: ixodidae) in northwestern Florida.

A 2-yr study was conducted in a northwestern Florida state park and recreation area to determine tick species composition, seasonal abundance, and spatial distribution. Risk of tick attachment to park visitors was also assessed relative to tick abundance from several habitats. Tick collections consisted of weekly cloth drag samples obtained from ground and emergent vegetation along a deer trail, a walking trail located in a 3-mo-old control burned area of forest, a nonburned walking trail, picnic area, and campsites. Sampling was conducted from February 1994 through February 1996. Ticks collected during the study were as follows in descending order of abundance: Ixodes scapularis Say, Amblyomma americanum (L.), A. maculatum Koch, and Dermacentor variabilis (Say). Significantly more (<0.05) adult ticks were recovered from vegetation compared with ground samples only for I. scapularis. Adult questing I. scapularis were collected from October through May (peak December). A. americanum adults were collected from March through August (peak May). Adult A. maculatum were collected during August and September and adult D. variabilis were collected July and August. Larval and nymphal stages of questing A. americanum were collected from June through November (peak July) and February through October (peak September), respectively. A. maculatum nymphs were collected from February through April, June, and September (peak March). No nymphs of I. scapularis were collected. Only four larvae of D. variabilis were collected during the study, all during February 1995. No I. scapularis or A. maculatum larvae were collected from vegetation or ground drags. The greatest risk for tick attachment was in campsites where approximately 60% of all adult and nymphal host-seeking ticks were collected. The next greatest risk area was the walking trail located in the control burned area where an additional 28% of ticks were obtained.

Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins.

The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined. Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody. The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity. This region appears to be conserved in all of the structurally defined plant RIPs examined. Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue. By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs. In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope. Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.

Free energy determinants of binding the rRNA substrate and small ligands to ricin A-chain.

A continuum model is provided of the free energy terms that contribute to the molecular association of ricin A-chain (RTA) with the rRNA substrate and several small ligands. The model for RTA interactions with the RNA was taken from a previously proposed complex containing a 29-mer oligonucleotide hairpin (. Proteins 27:80-95), and models for the ligands were constructed from x-ray crystallographic structures. The calculated absolute free energies of complex formation for the RTA-RNA assembly and several single-residue substitutions are in good agreement with experimental data, given the approximations of evaluating the strain energy and conformational entropy. The free energy terms were found to resemble those of protein-protein complexes, with the net unfavorable electrostatic contribution offset by the favorable nonspecific hydrophobic effect. Decomposition of the RTA-RNA binding free energy into individual contributions revealed the electrostatic "hot" spots arising from charge-charge complementarity of the interfacial arginines with the RNA phosphate backbone. Base interactions of the GAGA loop structure dominate the hydrophobic complementarity. A linear-scaling model was parametrized for evaluating the binding of small ligands against the rRNA substrate and illustrates the free energy determinant required for designing specific RTA inhibitors.

Identifying the principal protective antigenic determinants of type A botulinum neurotoxin.

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned. Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc. However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.

Development of engineered vaccines effective against structurally related bacterial superantigens.

Staphylococcal and streptococcal superantigens are exotoxins that may be linked to many human pathologies involving impaired immune functions. Despite considerable sequence divergence, bacterial superantigens share extensive secondary and tertiary structure and use similar structural strategies to bind major histocompatibility complex class II receptors. We produced by site-directed mutagenesis of the conserved receptor-binding surfaces of the superantigens staphylococcal enterotoxins A and B. These vaccines protected immunized mice and rhesus monkeys from lethal toxic shock. In addition, antibodies produced against each superantigen recognized and neutralized distantly related superantigens. This antibody cross-reactivity was additive in that mixtures of superantigens used in immunization were more effective than single-component vaccines in protecting mice from challenges with individual or mixed superantigens. We conclude that an optimal combination of these genetically attenuated superantigen vaccines may protect against all structurally related superantigens.