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This paper deals with the problem of joint state and unknown input estimation for stochastic discrete-time linear systems subject to intermittent unknown inputs on measurements. A Kalman filter approach is proposed for state prediction and intermittent unknown input reconstruction. The filter design is based on the minimization of the trace of the state estimation error covariance matrix under the constraint that the state prediction error is decoupled from active unknown inputs corrupting measurements at the current time. When the system is not strongly detectable, a sufficient stochastic stability condition on the mathematical expectation of the random state prediction errors covariance matrix is established in the case where the arrival binary sequences of unknown inputs follow independent random Bernoulli processes. When the intermittent unknown inputs on measurements represent intermittent observations, an illustrative example shows that the proposed filter corresponds to a Kalman filter with intermittent observations having the ability to generate a minimum variance unbiased prediction of measurement losses.

State estimation of stochastic discrete-time linear systems subject to unknown inputs has been widely studied, but few works take into account disturbances switching between unknown inputs and constant biases. We show that such disturbances affect a networked control system subject to deception attacks on the control signals transmitted by the controller to the plant via unreliable networks. This paper proposes to estimate the switching disturbance from an augmented state version of the intermittent unknown input Kalman filter. The sufficient stochastic stability conditions of the obtained filter are established when the arrival binary sequence of data losses follows a Bernoulli random process.

OBJECTIVE: Inhibition of fibrin sheath formation by enoxaparin decreases catheter colonization. Fibrin-binding radioactive tracer and catheter weights quantify fibrin reduction. DESIGN: Controlled experimental study of central venous line colonization. SETTING: Animal laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Central venous lines were introduced into right external jugular veins of 254 animals in three groups: enoxaparin, Fibrimage, and catheter weight. The enoxaparin group (n = 196) received daily enoxaparin injections (n = 97) or catheter implantation only (n = 99); 176 received tail vein injections of Staphylococcus epidermidis on postoperative day (POD) 10. Twenty rats received saline injections as a control. On POD 13, catheters were removed and incubated in broth at 37 degrees C for 48 hrs. Turbid samples were plated. In the Fibrimage group (n = 39), 20 rats receiving enoxaparin were compared with 19 controls without enoxaparin; all received S. epidermidis injections on POD 10. Fibrimage, fibrin-binding radiolabeled tracer, was given 1 hr before catheter removal. In the weight group (n = 19), six rats received enoxaparin; 13 did not. All received injections of S. epidermidis on POD 10. MEASUREMENTS AND MAIN RESULTS: Positive plates underwent analytic profile index testing, ensuring correlation with inoculum. Results were compared using Fisher's exact or chi-square tests. Gamma counts were determined in the Fibrimage group. Catheter tip weights were recorded. Results from the Fibrimage and weight groups were compared using Student's t-test. The enoxaparin group had fewer catheters colonized (17 of 77) vs. no enoxaparin (42 of 99; p < .01). Pericatheter sheaths contained less fibrin compared with controls. Fibrimage group gamma counts were significantly decreased for the enoxaparin subgroup (x = 2244 counts per minute) vs. controls (x = 3767 counts per minute; p < .0002). The weight of catheter tips treated with enoxaparin (x = 39 mg) vs. controls (x = 90 mg) was also significantly decreased (p < .0001). CONCLUSIONS: Enoxaparin decreases the amount of fibrin surrounding central venous catheters. The incidence of catheter colonization decreases when the amount of fibrin within the pericatheter sheath decreases. [ABSTRACT FROM AUTHOR]

Abstract Background Polychlorinated biphenyls (PCBs) are ubiquitous industrial chemicals that persist in the environment and in human fatty tissue. PCBs are related to a class of compounds known as dioxins, specifically 2,3,7,8-TCDD (tetrachloro-dibenzodioxin), which has been implicated as a cause of altered sex ratio, especially in relation to paternal exposures. Methods In the 1960's, serum specimens were collected from pregnant women participating in the Child Health and Development Study in the San Francisco Bay Area. The women were interviewed and their serum samples stored at -20°C. For this study, samples were thawed and a total of eleven PCBs were determined in 399 specimens. Secondary sex ratio, or sex ratio at birth, was evaluated as a function of maternal serum concentrations using log-binomial and logistic regression, controlling for hormonally active medications taken during pregnancy. Results The relative risk of a male birth decreased by 33% comparing women at the 90th percentile of total PCBs with women at the 10th percentile (RR = 0.67; 95% CI, 0.48–0.94; p = 0.02), or by approximately 7% for each 1 μg/L increase in total PCB concentration. Although some congener-specific associations with sex ratio were only marginally statistically significant, all nine PCB congeners with < 30% of samples below the LOQ showed the same direction of association, an improbable finding under the null hypothesis. Conclusion Maternal exposure to PCBs may be detrimental to the success of male sperm or to the survival of male embryos. Findings could be due to contaminants, metabolites or PCBs themselves.

Abstract The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, affecting approximately 6.5 million older adults in the United States. Development of AD treatment has primarily centered on developing pharmaceuticals that target amyloid-β (Aβ) plaques in the brain, a hallmark pathological biomarker that precedes symptomatic AD. Though recent clinical trials of novel drugs that target Aβ have demonstrated promising preliminary data, these pharmaceuticals have a poor history of developing into AD treatments, leading to hypotheses that other therapeutic targets may be more suitable for AD prevention and treatment. Impaired brain energy metabolism is another pathological hallmark that precedes the onset of AD that may provide a target for intervention. The brain creatine (Cr) system plays a crucial role in maintaining bioenergetic flux and is disrupted in AD. Recent studies using AD mouse models have shown that supplementing with Cr improves brain bioenergetics, as well as AD biomarkers and cognition. Despite these promising findings, no human trials have investigated the potential benefits of Cr supplementation in AD. This narrative review discusses the link between Cr and AD and the potential for Cr supplementation as a treatment for AD., (© 2023 The Authors.)

Introduction: A total of 100 nasal swabs were collected from healthy horses in Switzerland between January 2020 and August 2020. The samples were taken from horses at 40 different stables in 12 different cantons and screened for both methicillin-resistant (MRSA) and methicillin-susceptible S. aureus (MSSA) using selective agar plates. S. aureus were tested for antibiotic susceptibility by measurement of the minimal inhibitory concentration (MIC) and for virulence factors, antibiotic resistance genes and phylogenetic characteristics using whole genome sequence analysis. Ten horses were found to be positive (10 %, CI: 95 %, 0,0552 - 0,1744) for S. aureus, and four of them harboured MRSA (4 %, CI: 95 %, CI: 1,5 % - 9 %). The MRSA were detected in horses from three different stables in the same region of one canton and MSSA were detected in horses from five different cantons. All the MRSA isolates were genetically related (ST398-t011-IVa), while the MSSA were diverse (ST1-t127/t398/t1508, ST816-t1294, ST133-t1403, ST30-t012). MRSA showed resistance to penicillin (blaZ), cefoxitin (mecA), trimethoprim (dfrK), gentamicin, kanamycin (aac(6')-Ie - aph(2'')-Ia), and tetracycline (tet(M)). MSSA were resistant to either none or one of the antibiotics tested like penicillin (blaZ) and erythromycin (erm(T)). Virulence genes were more abundant in MSSA than in MRSA. This study provides first insight into the prevalence and type of S. aureus in healthy Swiss horses and reveals a source of strains, which may cause infections in both horses and humans.

Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by clinical decline in memory and other cognitive functions. A classic AD neuropathological hallmark includes the accumulation of amyloid-β (Aβ) plaques, which may precede onset of clinical symptoms by over a decade. Efforts to prevent or treat AD frequently emphasize decreasing Aβ through various mechanisms, but such approaches have yet to establish compelling interventions. It is still not understood exactly why Aβ accumulates in AD, but it is hypothesized that Aβ and other downstream pathological events are a result of impaired bioenergetics, which can also manifest prior to cognitive decline. Evidence suggests that individuals with AD and at high risk for AD have functional brain ketone metabolism and ketotherapies (KTs), dietary approaches that produce ketone bodies for energy metabolism, may affect AD pathology by targeting impaired brain bioenergetics. Cognitively normal individuals with elevated brain Aβ, deemed "preclinical AD," and older adults with peripheral metabolic impairments are ideal candidates to test whether KTs modulate AD biology as they have impaired mitochondrial function, perturbed brain glucose metabolism, and elevated risk for rapid Aβ accumulation and symptomatic AD. Here, we discuss the link between brain bioenergetics and Aβ, as well as the potential for KTs to influence AD risk and progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Taylor, Sullivan, Keller, Burns and Swerdlow.)

Introduction: The prevalence of methicillin-resistant Macrococcus spp. in calves and pigs at slaughterhouses and in retail beef and pork meat was determined using samples taken in 2019 within the framework of the national monitoring of methicillin-resistant Staphylococcus aureus in food producing animals in Switzerland. The isolates were submitted to antimicrobial susceptibility testing of 19 antibiotics and to molecular techniques (e.g. PCR, microarray, WGS) for the identification of resistance genes, elements containing the methicillin resistance genes mec and sequence type (ST). Methicillin-resistant Macrococcus spp. (M. caseolyticus (n=38), M. bohemicus (n=4) and Macrococcus spp. (n=2)) were isolated in 40 of 299 nasal swabs from calves representing a prevalence of 13,38 % (95 % CI, 9,98 % - 17,70 %), and in four of 303 nasal swabs from pigs [1,32 % (95 % CI, 0,36 % - 3,35 %)]. One of 311 samples of Swiss pork meat contained a Macrococcus sp. [0,32 % (95 % CI, 0,01 % - 1,78 %)], and four of 309 beef meat samples (260 domestic and 49 imported) contained M. caseolyticus [1,29 % (95 % CI, 0,35 % - 3,28 %)]. The M. caseolyticus strains belonged to diverse STs, with ST21 being the most common in both pigs and calves. The mecD gene was located on Macrococcus resistance island mecD (McRImecD) in 42 strains and on staphylococcal cassette chromosome mec (SCCmecD) in three strains, while mecB was found on plasmids in four strains. In addition to resistance to β-lactams, the strains also exhibited resistance to tetracycline (n=17; tet(L), tet(K), tet(M)), streptomycin (n=13; str, ant(6)-Ia, rpsL mutation [K56R in ribosomal protein S12]), kanamycin (n=10; aac(6')-Ie - aph(2'')-Ia, aph(2')-Ib, aph(2')-Ic, ant(4')-Ia), clindamycin (n=9; erm(B), erm(45)), erythromycin (n=9; erm(B), msr(G), erm(45)), fusidic acid (n=9; fusC) and gentamicin (n=1; aac(6')-Ie - aph(2'')-Ia). This study represents the first national prevalence study of methicillin-resistant Macrococcus spp. in pigs, calves, pork and beef meat in Switzerland and revealed a reservoir of genetically diverse strains carrying several resistance traits.

Gram-positive coccoid bacteria were isolated from the nasal cavities of pigs and calves as well as from axillar and inguinal skin regions of pigs. Phylogenetic analysis of seven strains based on complete genome, 16S rRNA, hsp60 , dnaJ , rpoB and sodA gene sequences and MALDI-TOF MS profiles revealed that they belonged to the genus Macrococcus with the closest relatedness to Macrococcus canis , Macrococcus caseolyticus subsp. caseolyticus and Macrococcus caseolyticus subsp. hominis . DNA relatedness of the type strain JEK37 T with the type strains of M. canis , M. caseolyticus subsp. caseolyticus and M. caseolyticus subsp. hominis was 23.4, 23.1 and 23.0 % by digital DNA-DNA hybridization and 80.39, 80.45 and 80.87 % by average nucleotide identity (ANI) calculations, confirming that they do not belong to the same species. The DNA G+C content of JEK37 T was 35.65 mol%. The novel strains can be differentiated from M. canis KM 45013 T by the ability to fermentate d-ribose and by the absence of DNAase production and haemolysis, from M. caseolyticus subsp. caseolyticus CCUG 15606 T by the ability to fermentate sucrose and from both species by the inability to grow in 9 and 12% NaCl. They differ from M. caseolyiticus subsp. hominis by the presence of α-glucosidase. The most common fatty acids of JEK37 T were C 14 : 0 , C 18 : 1  ω9 c and C 18 : 0 . Known polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, aminolipid, aminoglycolipid, aminophospholipid, glycolipid and phospholipid. Cell-wall peptidoglycan of JEK37 T was of type A3α l-Lys-Gly 2 -L-Ser-Gly (similar to A11.3) and the respiratory quinolone was menaquinone 6. Based on their genotypic and chemotaxonomic characteristics, these strains represent a novel species of the genus Macrococcus , for which we propose the name Macrococcus armenti sp. nov. The type strain is JEK37 T (=DSM 112712 T =CCOS 1982 T ).

Pressure-mediated reflection spectroscopy (RS) measures skin carotenoid content (SCC). Continued validation of this method is pertinent to validate its use as an objective measure of fruit and vegetable intake. This study aimed to assess relationships between SCC scores and self-reported dietary carotenoid intake from 3-day food records in community dwelling older adults. This was a cross-sectional analysis of baseline data among cognitively normal older adults (n=95) participating in the Nutrition Interventions for Cognitive Enhancement (NICE) study. Food-derived vitamin A (R=0.24, p<0.05) and food plus supplemental vitamin A (R=0.29, p<0.01) were correlated with SCC. All food-derived carotenoids (R: 0.20-0.39, p<0.05) except beta-cryptoxanthin and lycopene were correlated with SCC. Lutein + zeaxanthin from food more strongly correlated with SCC (R=0.38, p<0.001) than combined food plus supplemental intake (R=0.31, p=0.002). Correlations for total fruit and SCC (R=0.23, p=0.02), total vegetable and SCC (R= 0.31, p=0.002), and combined total fruit and vegetable and SCC (R=0.35, p<0.001) were significant; no sub-categories of fruit or vegetables except dark green vegetables (Rho=0.4, p<0.001) had significant correlations with SCC. The current study demonstrates RS-derived SCC scores correlate with 3-day self-reported intakes of F/V and certain carotenoids in community dwelling older adults., Competing Interests: Statement of Potential Conflict of Interest No potential conflict of interest reported by the authors.

BACKGROUND Statewide interventions are critical to meeting the goals of the National HIV/AIDS Strategy in this country. In 2012, the North Carolina Division of Public Health developed the North Carolina State Bridge Counselor program to improve linkage to and reengagement in care for newly diagnosed persons and persons living with HIV who were out-of-care. METHODS We reviewed the planning process for the North Carolina State Bridge Counselor program, which involved a review of existing strengths-based counseling models for persons living with HIV, implementation of these models, and communication strategies with other providers. State bridge counselor responsibilities were delineated from the role of disease intervention specialists while retaining the fieldwork capability of disease intervention specialists to conduct outreach and provide services for persons living with HIV throughout the state. RESULTS Program implementation required extensive planning with stakeholders, incorporation of strengths-based counseling models, development of performance standards, and utilization of CAREWare, an HIV care software program to document referrals and data-sharing between state bridge counselors and clinics. By the end of 2014, state bridge counselor services were provided to approximately 60 of the 400 persons living with HIV (15%) who are diagnosed each quarter in North Carolina, with increasing utilization of the program. LIMITATIONS We assessed the development of this intervention specific to the North Carolina Division of Public Health, which may limit its generalizability. However, the State Bridge Counselor program was implemented in both urban and rural areas throughout the state, which increases its applicability to different public health programs throughout the country. CONCLUSION We demonstrated that a statewide State Bridge Counselor program for linkage and reengagement activities can be implemented by leveraging existing infrastructures, electronic medical records, HIV care networks, and fieldwork activities., (©2018 by the North Carolina Institute of Medicine and The Duke Endowment. All rights reserved.)

Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP) and four distinct constituents of the mobile genome (mobilome): a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a common ancestor.

Background: The influence of psychotherapy on neurocognition in post-traumatic stress disorder (PTSD) has not been examined methodically. This is despite evidence that pre-treatment learning and memory has been associated with treatment success and that executive function theories emphasize weak executive functions (especially inhibition/switching) are associated with PTSD., Objectives: To determine (1) if higher pre-treatment learning/memory, inhibition/switching, or both predict treatment success; and (2) if treatment success is associated with specific improvement in inhibition/switching and not learning/memory or working memory, another aspect of executive function., Methods: Pre-treatment neurocognition and neurocognitive changes (inhibition/switching, learning/memory, working memory) were examined in female veterans with PTSD. They were evaluated before and after 16-weeks of group psychotherapy for PTSD that included three counterbalanced modules (cognitive restructuring therapy, exposure therapy, skills training) with fidelity checks for therapist adherence., Results: Only pre-treatment learning/memory predicted better treatment outcome. Treatment success was associated with improvement in inhibition/switching only, even after controlling for mild traumatic brain injury, and changes in depressive symptoms, working memory, and learning/memory., Conclusions: Our finding that learning/memory predicted treatment success is consistent with previous studies. We extended these studies by showing that the effect was restricted to learning/memory, which is contrary to the executive function theory of PTSD. In contrast, the fact that only inhibition/switching significantly improved with better treatment success is consistent with its potential importance in maintaining PTSD symptoms. Future research should determine whether inhibition/switching abilities are a risk for development and maintenance of PTSD or whether such abilities have a broader reciprocal relationship with PTSD symptom change. (JINS, 2016, 22, 643-651).

Neurocognitive problems are common with posttraumatic stress disorder (PTSD) and are important to understand because of their association with the success of PTSD treatment and its potential neural correlates. To our knowledge, this is the first neurocognitive study in an all-female U.S. veteran sample, some of whom had PTSD. We examined neurocognitive performance and assessed whether learning deficits, common in PTSD, were associated with executive functioning. Veterans with PTSD (n = 56) and without (n = 53) were evaluated for psychiatric and neurocognitive status. The PTSD group had a lower estimated IQ (d = 0.53) and performed more poorly on all neurocognitive domains (d range = 0.57-0.88), except verbal retention (d = 0.04). A subset of the 2 groups that were matched on IQ and demographics similarly demonstrated poorer performance for the PTSD group on all neurocognitive domains (d range = 0.52-0.79), except verbal retention (d = 0.15). Within the PTSD group, executive functioning accounted for significant variance in verbal learning over and above IQ and processing speed (ΔR(2) = .06), as well as depression (ΔR(2) = .07) and PTSD severity (ΔR(2) = .06). This study demonstrated that female veterans with PTSD performed more poorly than females without PTSD on several neurocognitive domains, including verbal learning, processing speed, and executive functioning. Replication of these results using a control group of veterans with more similar trauma exposure, history of mild traumatic brain injury, and psychiatric comorbidities would solidify these findings., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Laparoscopic ventral hernia repair requires placement of an intraperitoneal prosthetic. Composite mesh types have been developed to address the shortcomings of standard meshes. The authors evaluated the host reaction to intraperitoneal placement of a novel composite material., Materials and Methods: A comparison of an innovative polypropylene/polylactide composite mesh was made to parietex composite (PCO), Proceed, and DualMesh. Eighteen meshes per group were implanted on intact peritoneum in New Zealand white rabbits. The main outcome measures included the formation of visceral adhesions, adhesion tenacity, tensiometric measurements, and histological analysis. Evaluations of adhesions were made at 1, 4, and 16 weeks using a 2-mm minilaparoscopy., Results: There were no significant differences in the mean adhesion scores between the composite mesh types at week 1 (P = .15) and week 16 (P = .06). At 4 weeks, PCO had significantly fewer adhesions when compared with the other 3 mesh types (P = .02). Adhesion tenacity was also equivalent within the group at 16 weeks (P = .06). Tensiometry and histological analysis revealed no statistically significant differences between the mesh types., Conclusions: Four different composite mesh types had equivalent intra-abdominal soft tissue attachments in a rabbit model after a 16-week implantation period. PCO demonstrated the lowest mean adhesion score of each mesh type. Each mesh exhibited equivalent stiffness and energy to failure after explantation. The 4 composite mesh types demonstrated the successful formation of a neoperitoneum and comparable host biocompatibility as evidenced by similar degrees of inflammation.

Hyperimmune monovalent antitoxins to botulinum neurotoxin serotypes A and B have been produced by immunizing horses with newly developed formalin toxoids. After primary immunization, horses developed acceptable prophylactic antibody titers (1-5 IU/mL). Three horses received additional toxoid booster injections to induce hyperimmune antibody titers with antitoxin-A and antitoxin-B titers reaching peaks of approximately 2000 IU/mL and 150-625 IU/mL, respectively. Titers were quantified throughout the process by antigen-capture ELISA and by in-vivo neutralization. ELISA titers and neutralization titers correlated (R² ∼0.62-0.92), however, unique correlations between in-vitro and in-vivo titers were observed for each horse. Monovalent antitoxin pools were made by combining plasma that had been collected twice via plasmaphoresis several months after primary immunization. Neutralizing units were established for each pool relative to the current US and WHO reference standards. Titers were determined at the L(+)/10 and L(+)/40 toxin dose for Toxin types A and B, respectively, and U.S. and international units were assigned to each monovalent antitoxin. Avidity of the new Anti-A pool was equivalent to the WHO Anti-A reference at the L(+), L(+)/10 and L(+)/30 dose. Each monovalent plasma pool failed to cross-neutralize other botulinum neurotoxin serotypes indicating a high degree of specificity of each antitoxin for the toxin serotype used during immunization., (Published by Elsevier Ltd.)

Background: A recent series detailing thoracoscopic repair of esophageal atresia with tracheoesophageal fistula (EA/TEF) reported lower complication rates compared with historic controls. This study provides a contemporary cohort of patients repaired via thoracotomy for comparison with the recent large multi-institutional thoracoscopic series., Methods: Records of patients with EA/TEF between 1993 and 2008 were reviewed. Attention was focused on demographics and complications including anastomotic leak, recurrent fistulae, stricture formation, and need for fundoplication., Results: Seventy-two patients underwent repair of EA/TEF via thoracotomy. Complication rates in the current series compared with the thoracoscopic series were anastomotic leak, 2.7% versus 7.6%; recurrent fistulae, 2.7% versus 1.9%; stricture, 5.5% versus 3.8%; and need for fundoplication, 12% versus 24%. Differences in complication rates did not reach statistical significance. Two children in this cohort developed mild scoliosis attributed to congenital vertebral anomalies, neither of whom required intervention., Conclusions: Thoracoscopic repair of EA/TEF yielded complication rates similar to this contemporary series; however, trends toward increased anastomotic leaks and greater need for fundoplication were noted. No musculoskeletal sequelae were directly attributable to thoracotomy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Laparoscopic expertise increases the volume of adrenalectomies at referral centers. We report our 10-year experience with laparoscopic adrenalectomy. All laparoscopic adrenalectomies at a single institution were prospectively recorded in a surgical outcomes database. Patient demographics, operative/perioperative outcomes, and adrenal pathology were reviewed retrospectively. From March 1999 through July 2009, 154 laparoscopic adrenalectomies were performed in 150 patients. Average patient age was 49.9 years (range 15-82); mean body mass index was 31.1 kg/m(2) (range 17-56). Pathologic diagnoses included hyperaldosteronism (n = 69), nonfunctional adenoma (n = 28), pheochromocytoma (n = 23), hypercortisolism (n = 14), malignancy (primary n = 3, metastasis n = 9), and cyst (n = 4). Seventy-three per cent (n = 110) occurred on the left, 23 per cent (n = 35) on the right, 2.6 per cent (n = 4) bilateral, and 0.6 per cent (n = 1) as extra-adrenal. The average tumor measured 3.6 cm (range 0.4-12). The average operative time was 156 minutes (range 62-409), the mean estimated blood loss was 60 mL (range 10-400), and mean American Society of Anesthesiologists score was 2.6 (range 1-4). Three operations (0.2%) were converted to open. Three patients (0.2%) experienced perioperative complications (respiratory failure, urinary tract infection, line sepsis, and readmission within 30 days). The average length of stay was 3.4 days (range 1-44) and mean follow-up was 96.9 days (5-2567). No wound-related complications or deaths occurred. Pathologic diagnosis was not associated with a particular side or development of a complication (P > 0.5). Patients with pheochromocytomas had the longest operative times, highest estimated blood loss, and highest American Society of Anesthesiologists scores (218.2 minutes, 128 mL, 3.0; P < 0.004). Laparoscopic adrenalectomy is safe and effective. Removal of pheochromocytomas is more challenging and may be more appropriate for referral to a specialized center for optimal outcomes.

Background: Coating prosthetic for hernia repair with a patient's own cells could improve biocompatibility by decreasing inflammation and adhesion formation and by increasing tissue ingrowth and resistance to infection. The objective of this study was to prove the feasibility of prosthetic coating with stem cells and to assess its resistance to adhesion formation when implanted in an animal model., Methods: Adult Lewis rat bone marrow stem cells were harvested and cultured. Stem cells were then implanted on three different prosthetics. The prosthetic with the best stem cell adherence was implanted intraperitoneally into six adult rats. Untreated prosthetic was implanted in control animals (n = 12). After 2 weeks, intra-abdominal adhesions were graded using an adhesion scoring scale by two surgeons who were blinded to the animal group. Data were analyzed using the Wilcoxon rank-sum test., Results: Stem cells demonstrated the best adherence and growth on polyglactin prosthetics. After implantation, the stem cell-coated polyglactin prosthetic had <25% of its surface area covered with adhesions in five (83%) samples, whereas the control polyglactin group had only one sample (8.3%) with <25% adhesions, and seven of its samples (58.3%) had >50% surface area adhesions (p < 0.05)., Conclusions: The feasibility of hernia prosthetic coating with stem cells was demonstrated. Furthermore, stem-cell coated polyglactin prosthetic exhibited improved biocompatibility by decreasing adhesion formation in an animal model. Further study is needed to determine the factors that promote stem cell adherence to prosthetics and the in vivo prosthetic biomechanics after stem cell coating. This work is underway in our laboratory.

Background: Effectiveness of acellular human dermis (AHD) as an alternative to synthetic mesh in contaminated fields has been described. Cellular migration after implantation and corresponding strength of attachment is not well documented. Our aim is to correlate AHD vascularization, fibroblast migration, and strength of attachment with presence of inflammatory cells in clean and contaminated fields., Materials and Methods: Lewis rats were randomized to a control and three experimental groups. AHD was placed as an onlay over the intact abdominal wall. Experimental groups (n=72) were exposed to Staphylococcus aureus at 1 x 10(4), 1 x 10(5), or 1 x 10(6) by direct application; controls (n=12) were not exposed. At 5 and 28 d, abdominal walls were explanted and tissue ingrowth assessed via tensiometry measuring energy (E) and max stress (MS) at the AHD-tissue interface. Vascularity, fibroblast migration, and inflammatory cell migration were compared using light microscopy., Results: Shear strength reported as energy and max stress were significantly greater at 28 versus 5 d in all experimental groups, remaining unchanged in controls. Plasma cells and histiocytes significantly increased in all groups; macrophages increased in experimental groups only. Vascular ingrowth increased significantly in all groups; fibroblast migration was greater in controls and 1 x 10(6) exposed group only., Conclusions: Contamination of AHD results in inflammatory cell influx and a surprising increase in shear strength. Interestingly, shear strength does not increase without contamination. Inflammation stimulates vascular ingrowth, but not equally significant fibroblast migration. Longer survivals are required to determine if energy and max stress of controls increase, and fibroblast migration follows vascular ingrowth., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: Studies have shown donor and recipient outcomes to be equivalent for laparoscopic donor nephrectomy (LDN) and open donor nephrectomy. In the past, LDN has been avoided in the procurement of the right kidney or organs with multiple arteries. This study compares procurement of right and left kidneys as well as procurement of single- and multiple artery organs., Methods: A review of all LDNs at a single institution between August 2000 and December 2007 was performed. The data included estimated blood loss (EBL), need for transfusion, operative time, warm ischemia time, length of hospital stay (LOS), and delayed graft function. Arterial supply was assessed using renal arteriogram or computed tomographic (CT) angiography. Outcomes for multiple versus single artery and left versus right LDN were compared. Student's t-test and chi-square test were used for statistical comparison., Results: A total of 230 LDNs were performed. Multiple arteries were present in 37 donors. The right kidney was procured from 36 donors. No significant difference in EBL, transfusions, operative time, or LOS was noted between multiple and single or right and left LDNs. Warm ischemia time was significantly longer for multiple arteries (mean, 83 s) than for single arteries (mean, 63 s; p = 0.007), and for right kidneys (mean, 86 s) than for left kidneys (mean, 62 s; p = 0.001). No significant difference in delayed graft function was seen in the comparison of multiple (21.6%) and single (11.4%) artery organs (p = 0.11) or of right (13.9%) and left (12.9%) kidneys (p = 0.79)., Conclusions: The presence of multiple arteries or the need to procure the right kidney does not affect the operative outcome of laparoscopic donor nephrectomy. Warm ischemia time may be greater for these groups, but this does not result in delayed allograft function. The laparoscopic approach should be the standard of care even when expansion of the donor pool includes organs with multiple arteries and procurement of the right kidney.

The Kansas Geological Survey (KGS) developed a semianalytical solution for slug tests that incorporates the effects of partial penetration, anisotropy, and the presence of variable conductivity well skins. The solution can simulate either confined or unconfined conditions. The original model, written in FORTRAN, has a text-based interface with rigid input requirements and limited output options. We re-created the main routine for the KGS model as a Visual Basic macro that runs in most versions of Microsoft Excel and built a simple-to-use Excel spreadsheet interface that automatically displays the graphical results of the test. A comparison of the output from the original FORTRAN code to that of the new Excel spreadsheet version for three cases produced identical results.

Background: Minimally invasive surgical techniques have become the preferred method for live donor nephrectomy (DN) in many centers. We compared our experience with laparoscopic and open DN in a single institution., Methods: Data for 266 consecutive live DNs were collected. Demographic, intraoperative, and postoperative data were compared., Results: A total of 199 hand-assisted laparoscopic (HAL) DNs, 18 totally laparoscopic (TL), and 49 open DNs were performed. Laparoscopic DN was associated with a shorter operative time (p < 0.013), less blood loss (p < 0.0001), and shorter hospital stay (p < 0.0001) than open DN. Warm ischemia time was less for HAL versus TL DN (59.9 vs. 90.0 seconds; p < 0.0001). Compared with open DN, laparoscopic patients had fewer complications (p < 0.03), fewer wound infections (p < 0.004), less wound paresthesias (p < 0.0009), and fewer complaints of chronic incisional pain (p < 0.0001). Delayed graft function during the first 24 h postoperatively was significantly less for the laparoscopic DN versus the open cases (12.9% vs. 30.4%; p = 0.003), but the need for hemodialysis for the recipient was similar between groups (6.9% vs. 5%; p = not significant)., Conclusions: Laparoscopic DN resulted in less blood loss, reduced operative time, and shorter hospital stay than open DN. Hand-assisted laparoscopic DN has the potential to decrease warm ischemia time for renal allografts. Donors managed laparoscopically had fewer complications, significantly less wound-related morbidity, and less delayed graft function than patients who underwent open DN.

Background: Nonalcoholic fatty liver disease is associated with morbid obesity. Liver biopsy is the reference standard for the diagnosis of nonalcoholic fatty liver disease. It is unclear whether the macroscopic liver appearance correlates with the histopathologic findings. Our objective was to determine the relationship between the intraoperative liver appearance and the histopathologic findings during laparoscopic bariatric surgery at a tertiary medical center., Methods: Data were prospectively collected from 108 consecutive patients undergoing laparoscopic bariatric surgery with routine intraoperative liver biopsy. An intraoperative liver visual score was recorded according to the size, tan-speckling, and contour. The liver histologic findings were categorized into 3 groups: (1) normal; (2) bland steatosis; and (3) nonalcoholic steatohepatitis (NASH). The liver visual score was compared with the liver histologic findings. A recorded video of the liver was regraded at a later date to determine observer agreement., Results: The prevalence of NASH was 23% (n = 25). Of the 108 patients, 48% with NASH had normal-appearing livers and accounted for 24% (n = 12) of the 50 normal-appearing livers. A similar proportion of NASH cases was found in all 3 visual categories. Furthermore, no relationship was found between the number of abnormal visual cues and the liver histologic findings (P = .23). No complications were directly attributable to liver biopsy. The kappa values for intraobserver and interobserver agreement ranged from fair to almost perfect., Conclusion: NASH is common in the morbidly obese population. There does not appear to be a relationship between liver appearance and the histopathologic findings. Intraoperative liver biopsy is a safe and accurate method of diagnosing liver disease and should be considered in all morbidly obese patients undergoing abdominal surgery.

Background/purpose: We previously reported that a diet acidified with citric acid effectively reinforces gastric acid protection against bacterial colonization and translocation. In this study, our objective was to examine a biologically acidified formula hypothesized to be more physiologic than formula acidified with free acid. This study was Institutional Animal Care and Use Committee (IACUC) approved and designed to determine whether this diet is better tolerated and equally effective to acidification with citric acid against gut colonization and subsequent bacterial translocation in a premature infant rabbit model., Methods: A total of 89 rabbit pups born via cesarean delivery 1 day preterm were randomly assigned to 3 feeding groups: Pelargon Nestle at pH 4.55; NAN Nestle, a control diet at pH 7.0 with similar composition; and NAN Nestle acidified in the laboratory with citric acid at pH 4.55. Pups were gavage fed every 12 hours with Enterobacter cloacae challenges of 10 colony-forming units per milliliter of diet per feed and killed on day 3 of life. Lungs, liver, spleen, mesenteric lymph nodes, stomach, and cecum were cultured and quantitatively analyzed for target organism growth and statistically analyzed using chi(2) and Kruskal-Wallis tests., Results: Pelargon, compared to acidified NAN and NAN, significantly reduced the incidence of gastric colonization (15/33 [45%], 21/27 [78%], and 25/29 [86%], respectively; P < .01) and pulmonary colonization (10/33 [30%], 19/27 [70%], 21/29 [72%]; P < .01). Comparing the bacterial logs of colonized groups, the same benefit is observed in the lungs (0.77 +/- 1.22, 1.89 +/- 1.41, 2.12 +/- 1.47; P < .01). Gut colonization and bacterial translocation were equivalent between treatment groups (mesenteric lymph nodes: 10/33 [30%], 11/27 [40%], 8/29 [27%]; spleen: 10/33 [30%], 7/27 [26%], 8/29 [27%]; liver: 10/33 [30%], 6/27 [22%], 9/29 [31%]; cecum: 33/33 [100%], 27/27 [100%], 29/29 [100%])., Conclusion: Biologically acidified formula demonstrated superior protection against pulmonary and gastric colonization compared to normal pH and diets acidified with free acid. Its effects may potentially reduce clinical pulmonary infection.

Antigenicities of several formalin-detoxified botulinum neurotoxin preparations were measured by inhibition and sandwich enzyme-linked immunosorbent assay (ELISA), and immunogenicity was studied in mice. The toxoids were derived primarily from the serotype A 150-kDa neurotoxin protein, while one toxoid was derived from the naturally occurring 900-kDa toxin-hemagglutinin complex. Antigenicity was severely compromised in two commercially available toxoids. A variety of new toxoids were synthesized in-house by optimizing formaldehyde reaction conditions. Three of the resulting toxoids were found to be antigenically identical to the native toxin, as measured by inhibition ELISA, in spite of showing a reduction of toxicity by more than 100,000-fold. Sandwich ELISAs indicated that the in-house toxoids were two- to threefold less antigenic than the neurotoxin compared to commercial toxoids, which were about 100-fold less antigenic. Mice were immunized twice, on day 0 and day 14. By day 28, relatively high toxin-specific immunoglobulin G (IgG) titers were detected in animals that had received any of the in-house toxoids, with greater than 99% being IgG1 and the remainder being IgG2. These immunized mice remained asymptomatic after being challenged with 50 to 1,000,000 50% lethal dose (LD(50)) units of the 900-kDa neurotoxin. In contrast, animals immunized with several different batches of commercially available toxoids did not develop measurable toxin-specific antibody titers. However, these mice survived neurotoxin challenges with 2 LD(50) units but died when challenged with 6 LD(50) units. Neutralizing titers measured from pools of sera generated with the in-house toxoid preparations ranged from 2.5 to 5 U/ml. In terms of predicting immunogenicity, inhibition ELISAs comparing each formalin toxoid to the parent toxin provided good insight for screening the new toxoids as well as for estimating their relative in vivo potencies. Inhibition ELISA data indicate that those toxoids that most closely resemble the native toxin are highly immunogenic and protective. The superior quality of these new toxoids makes them useful tools for continued use in ELISA development and for antitoxin production.

Chronic groin pain is the most frequent long-term complication after inguinal hernia repair affecting up to 34 per cent of patients. Traditional surgical management includes groin exploration, mesh removal, and neurectomy. We evaluate outcomes of a combined laparoscopic and open approach to chronic pain after inguinal herniorrhaphy. All patients undergoing surgical exploration for chronic pain after inguinal herniorrhaphy were analyzed. In most, the operation consisted of mesh removal (open or laparoscopic), neurectomy, and placement of mesh in the opposite location of the first mesh (laparoscopic if the first was open and vice-versa). Main outcome measures included pain status, numbness, and hernia recurrence. Twenty-one patients (16 male and 5 female) with a mean age of 41 years (22-51 years) underwent surgical treatment for unilateral (n = 18) or bilateral (n = 3) groin pain. Percutaneous nerve block was unsuccessful in all patients. Four had previous surgery for pain. There were no complications. With a minimum of 6 weeks follow-up, 20 of 21 patients reported significant improvement or resolution of symptoms. A combined laparoscopic and open approach for postherniorrhaphy groin pain results in excellent patient satisfaction with minimal morbidity. It may be the preferred technique for the definitive management of chronic neuralgia after hernia repair.

The U.S. Environmental Protection Agency has established several methods to delineate wellhead protection areas (WHPAs) around community wells in order to protect them from surface contamination sources. Delineating a WHPA often requires defining the capture zone for a well. Generally, analytical models or arbitrary setback zones have been used to define the capture zone in areas where little is known about the distribution of hydraulic head, hydraulic conductivity, or recharge. Numerical modeling, however, even in areas of sparse data, offers distinct advantages over the more simplified analytical models or arbitrary setback zones. The systematic approach discussed here calibrates a numerical flow model to regional topography and then applies a matrix of plausible recharge to hydraulic conductivity ratios (R/K) to investigate the impact on the size and shape of the capture zone. This approach does not attempt to determine the uncertainty of the model but instead yields several possible capture zones, the composite of which is likely to contain the actual capture zone. A WHPA based on this composite capture zone will protect ground water resources better than one based on any individual capture zone. An application of the method to three communities illustrates development of the R/K matrix and demonstrates that the method is particularly well suited for determining capture zones in alluvial aquifers.

Background/purpose: Acidified diets are protective against intestinal bacterial colonization and translocation. Probiotic diets are designed to modulate the intestinal flora to enhance mucosal immunity. This study was designed to determine if formula acidified with live probiotic decreases bacterial gut colonization and translocation, and is equally tolerated as other acidified diets., Methods: One hundred twenty-eight rabbit pups delivered via cesarean section [cesarean delivery, cesarean birth, abdominal delivery] were randomly assigned to 4 feeding groups: NAN Nestle (control, pH 7.0), NAN acidified with citric acid (pH 4.55), biologically acidified Pelargon (pH 4.55), and NAN with live Lactococcus lactis culture (pH 4.2). Pups were gavage fed every 12 hours with Enterobacter cloacae challenges of 10 colony-forming units/mL per feed and killed on day of life 3. Lungs, liver, spleen, mesenteric lymph nodes (MLNs), stomach, and cecum were cultured and quantitatively analyzed for target organism growth. Results were analyzed using chi(2) tests., Results: NAN with live probiotic culture, when compared with Pelargon, acidified NAN, and NAN, significantly reduced the incidence of Enterobacter pulmonary colonization (P < .01), bacterial translocation (liver, P < .025; spleen and MLN, P < .05), and gastric and intestinal colonization (P < .001 for both)., Conclusion: Probiotic-fortified formula provides superior protection against pulmonary and gastrointestinal bacterial colonization and translocation compared with neutral and acidified formulas, and is equally tolerated.

Chronic wasting disease (CWD) in Wisconsin was first identified in February 2002. By April 2005, medial retropharyngeal lymph node (RLN) tissues had been examined from over 75,000 white-tailed deer for the presence of CWD by either immunohistochemical (IHC) staining for the prion protein associated with CWD (PrP(res)) or by using enzyme-linked immunosorbent assays with confirmation of positives by IHC staining and had been detected in 469 animals. Obex tissue was also available from 438 of the CWD-positive animals and was CWD positive by IHC staining in 355 (81%). To verify whether false-negative results were possible examining only RLN, both obex and RLN samples were examined for CWD by IHC staining from 4,430 of the white-tailed deer harvested from an area in Wisconsin where the overall deer CWD prevalence was approximately 6.2%. Two hundred and fourteen of the 269 positive deer (79.6%) had deposits of PrP(res) in both obex and lymphoid tissues, 55 (20.4%) had deposits only in lymphoid tissue, and there were no deer that had deposits only in obex.

Morbid obesity affects over 15 million people in the United States. Nonsurgical management produces sustained weight loss in less than 5% of patients. Despite associated comorbidities, less than 1% of obese patients seek surgical intervention. Less invasive procedures have been developed with varying success. The Endobarrier( trade mark) (GI Dynamics(trade mark), Watertown, MA) duodenal-jejunal bypass sleeve is a totally endoscopically delivered device designed to produce weight loss in the morbidly obese. We describe the first placement of a duodenal-jejunal bypass sleeve in a patient in the United States. A blinded, randomized, prospective clinical trial was approved by the Food and Drug Administration to evaluate safety and efficacy of a novel device for weight loss in the obese. The first patient enrolled was a 36-year-old woman with body mass index of 45.2. After informed consent, endoscopic placement of the device under general anesthesia was performed using fluoroscopy to confirm positioning. The device was placed without complications. At conclusion of the 3-month study period, the device was removed endoscopically. Total weight lost by the patient was 9.09 kg. Described herein is the first deployment of the duodenal-jejunal bypass sleeve in North America. The device is delivered in a totally endoscopic manner in morbidly obese patients. In our patient, total weight loss at 3 months was 9.09 kg. Continued follow-up and enrollment is ongoing to demonstrate patient safety and efficacy. Additional studies are being performed to elucidate mechanism of weight loss and future clinical applications of this device.

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

Background/purpose: Lipoblastoma/lipoblastomatosis, a rare benign pediatric tumor composed of embryonic fat, most often presents as a rapidly growing mass in children younger than 3 years. This benign tumor locally recurs up to 25% of the time. This retrospective review was done to emphasize problems with diagnosis, management, and the approach to follow-up care., Methods: Sixteen cases of pathologically proven lipoblastoma presenting to a single institution over a 16-year period (1988-2004) were reviewed retrospectively for clinical features and outcome., Results: There were 11 males and 5 females ranging in age from 18 days to 12.75 years. Preoperative diagnoses were accurate in only 3 cases. Two lesions recurred locally over a period of 10 months and 5 years. One lesion presented with local infiltration. Six lesions were right-sided, 5 were left-sided, and 5 were midline., Conclusions: It is important to consider lipoblastoma in the diagnosis of a rapidly enlarging fatty mass in children. Complete resection is the only definitive treatment and should not be delayed when impingement on surrounding structures is imminent. There is a tendency for these lesions to recur despite presumed complete excision. Therefore, follow-up for a minimum of 5 years is recommended.

Botulinum neurotoxins cause the disease botulism, which is characterized by prolonged muscle paralysis. In contrast, injections of low doses of purified botulinum neurotoxins do not cause systemic illness but produce localized muscle paralysis that is beneficial for treating several human medical disorders involving uncontrollable muscle contraction. Optimizing the therapeutic efficacy while diminishing adverse reactions requires precise knowledge of toxin potency as well as a clear understanding of how each toxin causes disease. A novel in vivo mouse assay has been used to correlate toxin dosage with the duration of muscle paralysis. Voluntary running activity performed by mice was proportional to the amount of toxin injected into the hind limbs and the subsequent rate of recovery over the ensuing days or weeks was a function of botulinum neurotoxin serotype A or B concentration. Botulinum neurotoxin A produced longer paralysis than botulinum neurotoxin B consistent with human observations. A third serotype, botulinum neurotoxin E, had the shortest duration of action, but unlike the other two toxins, dosage did not influence recovery time. Botulinum neurotoxin A recovery appeared biphasic with the initial phase about two-fold faster than the final phase. Over four weeks, muscle activity had gradually improved following the highest botulinum neurotoxin A dose, reaching about half of the normal running activity. Lower botulinum neurotoxin A doses led to incrementally faster and complete recovery. Persistence of maximum paralysis was exponentially related to botulinum neurotoxin A dosage, with a doubling of the paralysis time occurring with every 25% increase of the toxin concentration. In contrast, the rate of recovery from botulinum neurotoxin B was monophasic relative to toxin dosage and the duration of maximum paralysis was linear relative to dosage. Combinations of botulinum neurotoxin A and B and botulinum neurotoxin A and E were tested and shown to exacerbate paralysis compared with individually administered serotypes.

Severity of injuries from air-powered weapons can be underappreciated. Transformation of these weapons into toys makes them available to children. Our experience reveals the underestimated injury severity and emphasizes need for prompt trauma evaluation. Retrospective chart review of children sustaining air-gun injuries and evaluated at a single, pediatric hospital from 1991 to 2002 was performed. Medical record numbers were retrieved from a trauma data base. Data included age, weapon type, firing distance, injury site, radiographic studies, operative intervention, length of stay, and long-term disability. Ocular injuries were excluded secondary to known severity. All other injuries and treatments are described. Thirty-four children, average age 10 years +/- 3.3 years, sustained 35 injuries from 1991 through 2002. Twenty-one children required admission, 19 children required surgery, and 5 children experienced long-term disability. Average time to definitive care was 3 hours 12 minutes. Sites of injury included head, neck, chest, abdomen, and extremities. Average hospital stay was 4.3 days. In the pediatric population, air-gun injuries can be underestimated. Lack of collateral tissue damage makes wounds appear innocuous to unsuspecting medical personnel resulting in delayed care. During initial evaluation, injuries from air guns deserve the same respect as those caused by conventional firearms.

Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release. The toxin enters the neuron by binding to neuronal membrane receptor(s), being taken up into an endosome-like compartment, and penetrating the endosome membrane via a pH-dependent translocation process. Once within the synaptic cytoplasm, BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25, one of the SNARE proteins required for synaptic vesicle fusion. In this study, we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation. The results indicate that (1) uptake of both BoNT-A and -E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca2+ and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E. Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure, indicating that toxin endocytosis occurs with synaptic activity. Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM. IC50 values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes. Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC50 values of approximately 25 nM. Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action; however, addition of Con A after 40 min was no longer protective. In contrast, Con A inhibited, but did not prevent, translocation of BoNT-E even when added immediately after toxin exposure, indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A. This study demonstrates that uptake of both BoNT-A and -E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.

Botulinum neurotoxin B (BoNT/B) produces muscle paralysis by cleaving synaptobrevin/vesicle-associated membrane protein (VAMP), an 18-kDa membrane-associated protein located on the surface of small synaptic vesicles. A capillary electrophoresis (CE) assay was developed to evaluate inhibitors of the proteolytic activity of BoNT/B with the objective of identifying suitable candidates for treatment of botulism. The assay was based on monitoring the cleavage of a peptide that corresponds to residues 44-94 of human VAMP-2 (V51) following reaction with the catalytic light chain (LC) of BoNT/B. Cleavage of V51 generated peptide fragments of 18 and 33 amino acids by scission of the bond between Q76 and F77. The fragments and parent peptide were clearly resolved by CE, allowing accurate quantification of the BoNT/B LC-mediated reaction rates. The results indicate that CE is suitable for assessing the enzymatic activity of BoNT/B LC.

Previous work had demonstrated that organomercurial-mediated modification of two cysteine residues in the vesicular acetylcholine transporter (VAChT) from Torpedo californica inhibits binding of vesamicol. The cysteines are protected by acetylcholine and vesamicol (Keller et al. 2000. J. Neurochem. 74:1739-1748). Modified "cysteine 1" is accessible to glutathione from the cytoplasmic surface, whereas modified "cysteine 2" is not. Different organomercurials and aqueous environments were used here to characterize diffusion pathway(s) leading to the cysteines. para-Chloromercuriphenylsulfonate modifies VAChT much more slowly than do more hydrophobic p-chloromercuribenzoate and phenylmercury chloride. Permeabilization of vesicles with cholate detergent increases the rate of modification by p-chloromercuriphenylsulfonate. Permeabilization does not affect the ability of glutathione to reverse modification by p-chloromercuriphenylsulfonate. Higher ionic strength causes about four-fold increase in the rate of modification. The results suggest that hydrophobic and electrostatic barriers inhibit modification of Torpedo VAChT by negatively charged organomercurials and glutathione cannot reach cysteine 2 from either side of the membrane.

A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DV-associated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC.

Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137-142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca(2+). In this case, vesicular release is restored by increasing intracellular Ca(2+). These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca(2+).

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.

The role of proton binding sites in the vesicular acetylcholine transporter was investigated by characterization of the pH dependence for the binding of [3H]vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] to Torpedo synaptic vesicles. A single proton binds to a site with pKa 7.1 +/- 0.1, which is characteristic of histidine, to competitively inhibit vesamicol binding. The histidine-selective reagent diethylpyrocarbonate causes time-dependent inhibition of [3H]vesamicol binding with a rate constant only about 20-fold lower than for reaction with free histidine. Because its pH titration has a simple, ideal shape, this residue probably controls all pH effects in the transporter between pH 6-8. Inhibition of [3H]vesamicol binding by diethylpyrocarbonate was slowed by vesamicol but not acetylcholine, which binds to a separate site. The data suggest that a critical histidine with a pKa of 7.1 is unhindered when reacting with diethylpyrocarbonate. A conformational model for the histidine is proposed to explain why acetylcholine competes with protons but not with diethylpyrocarbonate. A conserved histidine in transmembrane helix VIII possibly is the histidine detected here.