The association between maternal colonization with Group B Streptococcus and infectious morbidity following transcervical Foley catheter-assisted labor induction.

OBJECTIVES: To determine whether maternal colonization with Group B Streptococcus increases the risk for infectious morbidity following transcervical Foley catheter-assisted cervical ripening. METHODS: A retrospective cohort study comparing infectious morbidity and other clinical outcomes by Group B Streptococcus colonization status between all women with singleton pregnancies who underwent Foley catheter-assisted cervical ripening labor induction at a single tertiary medical center during 2011-2021. Multivariable logistic regression explored the relationship between Group B Streptococcus colonization to adverse outcomes while adjusting for relevant clinical variables. RESULTS: A total of 4,409 women were included of whom 886 (20.1 %) were considered Group B Streptococcus carriers and 3,523 (79.9 %) were not. Suspected neonatal sepsis rate was similar between Group B Streptococcus carriers and non-carriers (5.2 vs. 5.0 %, respectively, p=0.78). Neonatal sepsis was confirmed in 7 (0.02 %) cases, all born to non-carriers. Group B Streptococcus carriers had a higher rate of maternal bacteremia compared to non-carriers (1.2 vs. 0.5 %, respectively, p=0.01). Group B Streptococcus colonization was independently associated with maternal bacteremia (adjusted odds ratio 3.05; 95 %CI 1.39, 6.66). CONCLUSIONS: Group B Streptococcus colonization among women undergoing Foley catheter-assisted cervical ripening does not seem to increase the risk for neonatal infection. However, higher rates of maternal bacteremia were detected.

A Novel Running Wheel Mouse Model for Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.

Antitoxin is currently the only approved therapy for botulinum intoxications. The efficacy of antitoxin preparations is evaluated in animals. However, while in practice antitoxin is administered to patients only after symptom onset, in most animal studies, it is tested in relation to time postintoxication. This may be attributed to difficulties in quantitating early botulism symptoms in animals. In the current study, a novel system based on high-resolution monitoring of mouse activity on a running wheel was developed to allow evaluation of postsymptom antitoxin efficacy. The system enables automatic and remote monitoring of 48 mice simultaneously. Based on the nocturnal activity patterns of individual naive mice, two criteria were defined as the onset of symptoms. Postsymptom treatment with a human-normalized dose of antitoxin was fully protective in mice exposed to 4 50% lethal doses (LD50s) of botulinum neurotoxin serotype A (BoNT/A) and BoNT/B. Moreover, for the first time, a high protection rate was obtained in mice treated postsymptomatically, following a challenge with BoNT/E, the fastest-acting BoNT. The running wheel system was further modified to develop a mouse model for the evaluation of next-generation therapeutics for progressive botulism at time points where antitoxin is not effective. Exposure of mice to 0.3 LD50 of BoNT/A resulted in long-lasting paralysis and a reduction in running activity for 16 to 18 days. Antitoxin treatment was no longer effective when administered 72 h postintoxication, defining the time window to evaluate next-generation therapeutics. Altogether, the running wheel systems presented herein offer quantitative means to evaluate the efficacy of current and future antibotulinum drugs.

Single- versus multiple-dose methotrexate in cesarean scar pregnancies management: treatment and reproductive outcomes.

PURPOSE: We aim to assess the outcome of the treatment of cesarean scar pregnancy (CSP) with single-dose methotrexate (MTX) versus multiple-dose MTX protocols. METHODS: A retrospective cohort study including two tertiary medical centers was conducted. All women diagnosed with CSPs between the years 2011 and 2019 that were initially managed with systemic MTX were included. Single-dose MTX practiced in one medical center was compared to multiple-dose MTX, practiced in the other medical center. RESULTS: The study cohort included 31 women in the single dose and 32 women in the multiple-dose MTX groups. Baseline characteristics did not differ between groups. The primary outcome occurred in 12 (38.7%) of the cases in the single-dose group and in 6 (18.8%) in the multiple-dose group (p = 0.083). The rate of conversion to surgical treatment was similar in both groups (4 vs. 5 in the single vs. multiple-dose groups, respectively, p = 0.758). There was no significant difference between the single- and the multiple-dose groups in the administration of blood products (16.1% vs. 3.1%, respectively, p = 0.104), total days of admission (18 ± 9.3 vs. 17 ± 12.8 days, respectively, p = 0.850), and readmission rate (32.3% vs. 21.9%, respectively, p = 0.353). Data regarding sequential pregnancies were available for 11 women in the single and 13 women in the multiple-dose MTX groups. There were no differences between the groups in rates of term deliveries, CSP recurrence, and abortions. CONCLUSION: Both single- and multiple-dose MTX treatment protocols offer high success rate with a relatively low complication rate in the treatment of CSP.

Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E.

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

Nonsurgical Management of Interstitial Pregnancies: Feasibility and Predictors of Treatment Failure.

STUDY OBJECTIVES: To describe the management of interstitial pregnancies in a tertiary medical center, identify factors associated with treatment failure, and report subsequent pregnancy outcome. DESIGN: Retrospective cohort study. SETTING: Department of Gynecology in a tertiary medical center. PATIENTS: All women who were admitted to and treated for interstitial pregnancy at our center between 2011 and 2019. INTERVENTIONS: The women were originally assigned to undergo expectant, medical, or surgical treatment. The women's background and clinical data were compared according to initial treatment modality. Nonsurgical (expectant and medical) management outcomes were analyzed to identify risk factors for treatment failure. Subsequent pregnancy outcomes were described separately. MEASUREMENT AND MAIN RESULTS: Thirty-seven cases of interstitial pregnancy were identified. There were high rates of pregnancy achieved by in vitro fertilization (45.9%) and a history of ipsilateral salpingectomy (43.2%) among these patients. At presentation, the mean age of the study cohort was 34.76 years, and the median ß-human chorionic gonadotropin level was 3853.0, and median gestational age was 7.0, respectively. The nonsurgical management success rate was 70.0%. Uterine rupture occurred during treatment in 5 cases (16.6%). Gestational sac diameter significantly affected treatment failure (p = .03), and a diameter >20 mm was observed in all cases of failed non-surgical treatment. Data on future fertility was available for 21 (58.3%) women: 13 (61.9%) had a subsequent pregnancy, 1 of which was a recurrent interstitial pregnancy. The median interpregnancy interval was 8.1 months, and all but 3 pregnancies reached third trimester and resulted in a live birth, with an overall cesarean delivery rate of 61.5%. None of the subsequent pregnancies were complicated by uterine rupture, and no serious adverse outcomes were noted in any of the subsequent intrauterine pregnancies that reached third trimester. CONCLUSION: Successful nonsurgical management of an interstitial pregnancy is feasible, although appropriate selection of cases is advised. A large gestational sac is a risk factor for treatment failure and should prompt surgical intervention. Subsequent pregnancies can generally be considered safe and with a favorable outcome.

Does antenatal steroids treatment in twin pregnancies prior to late preterm birth reduce neonatal morbidity? Evidence from a retrospective cohort study.

PURPOSE: To evaluate whether antenatal corticosteroids (ACS) administration during the late-preterm (LPT) period in twin pregnancies is associated with decreased rate of neonatal morbidity. METHODS: A retrospective cohort study including a total of 290 women with twin pregnancies resulting in live births of 580 neonates who delivered during LPT period between 2016 and 2018 at a tertiary medical center. Patients were allocated into two groups according to ACS exposure. Obstetric and neonatal outcomes were compared between the groups. Primary outcome was neonatal composite respiratory morbidity, defined as the occurrence of at least one of the followings: RDS, TTN, O2 requirement, CPAP use or mechanical ventilation. RESULTS: Patients exposed to ACS were older and more commonly complicated by gestational diabetes compared to the non-exposed group. Moreover, women exposed to ACS delivered earlier (35.6 vs. 36.3 weeks, P < 0.001) and more frequently by cesarean section (76.4% vs. 54.1%, P = 0.002) compared to the non-exposed group. The rate of composite respiratory morbidity did not differ between the groups. Nevertheless, neonates exposed to ACS had higher rates of neonatal intensive care unit (NICU) admission and hypoglycemia compared to neonates without prior ACS exposure (27.8% vs. 11.7%, P = 0.001; 49.3% vs. 27.1%, P < 0.001, respectively). Multivariable logistic regression revealed that gestational age at delivery was the sole independent risk factor for NICU admission, whereas late-preterm ACS exposure was the only risk factor for hypoglycemia. CONCLUSION: LPT-ACS administration in twin pregnancies complicated by LPT birth in our study did not reduce neonatal respiratory morbidity but was associated with higher rates of hypoglycaemia.

A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.

Botulinum neurotoxins (BoNTs), the most poisonous substances known in nature, pose significant concern to health authorities. The only approved therapeutic for botulism is antitoxin. While administered to patients only after symptom onset, antitoxin efficacy is evaluated in animals mostly in relation to time postintoxication regardless of symptoms. This is most likely due to the difficulty in measuring early symptoms of botulism in animals. In this study, a rabbit spirometry model was developed to quantify early respiratory symptoms of type E botulism that were further used as a trigger for treatment. Impaired respiration, in the form of a reduced minute volume, was detected as early as 18.1 ± 2.9 h after intramuscular exposure to 2 rabbit 50% lethal doses (LD50) of BoNT serotype E (BoNT/E), preceding any visible symptoms. All rabbits treated with antitoxin immediately following symptom onset survived. Postsymptom antitoxin efficacy was further evaluated in relation to toxin and antitoxin dosages as well as delayed antitoxin administration. Our system enabled us to demonstrate, for the first time, full antitoxin protection of animals treated with antitoxin after the onset of objective and quantitative type E botulism symptoms. This model may be utilized to evaluate the efficacy of antitoxins for additional serotypes of BoNT as well as that of next-generation anti-BoNT drugs that enter affected cells and act when antitoxin is no longer effective.

Pregnancy and Birth Outcomes Among Primiparae at Very Advanced Maternal Age: At What Price?

OBJECTIVES: In light of the potential physical and emotional costs to both woman and child, this study was conducted to assess pregnancy complications and birth outcomes in primiparae at very advanced maternal age (VAMA, aged ≥45) compared to younger primiparae. METHODS: Retrospective cohort study comparing 222 VAMA primiparae and a reference group of 222 primiparae aged 30-35, delivering at Sheba Medical Center from 2008 through 2013. RESULTS: VAMA primiparae were more likely than younger primiparae to be single, to have chronic health conditions, and higher rates of gestational diabetes mellitus (GDM), gestational-hypertension (GHTN) and preeclampsia-eclampsia. VAMA primiparae conceived mostly by oocyte donation. They were more likely to be hospitalized during pregnancy, to deliver preterm and by cesarean birth. Infants of VAMA primiparae were at greater risk for low birthweight and Neonatal Intensive Care Unit admission. There were no differences in outcomes between VAMA primiparae with or without preexisting chronic conditions, or between those aged 45-49 and ≥50. In multivariable analysis VAMA was an independent risk factor for GDM, GHTN and preeclamsia-eclampsia, with adjusted odds ratio of 2.38 (95 % CI 1.32, 4.29), 5.80 (95 % CI 2.66, 12.64) and 2.45 (95 % CI 1.03, 5.85); respectively. The effect of age disappeared in multiple pregnancies. CONCLUSIONS: Primiparity at VAMA holds a significant risk for adverse pregnancy and birth outcomes. The absence of chronic medical conditions or the use of a young oocyte donor does not improve these outcomes. Multiple pregnancies hold additional risk and may diminish the effect of age. Primiparity at an earlier age should be encouraged.

Early, Real-Time Medical Diagnosis of Botulism by Endopeptidase-Mass Spectrometry.

Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure.

Expression, purification and characterization of the receptor-binding domain of botulinum neurotoxin serotype B as a vaccine candidate.

The receptor-binding domain of botulinum neurotoxins (the HC fragment) is a promising vaccine candidate. Among the HC fragments of the seven BoNT serotypes, the expression of HC/B in Escherichia coli is considered especially challenging due to its accumulation as a non-soluble protein aggregate. In this study, the effects of different parameters on the expression of soluble HC/B were evaluated using a screening assay that included growing the bacterium at a small scale, a chemical cell lysis step, and a specific ELISA. The highest soluble HC/B expression levels were obtained when the bacterium E. coli BL21(DE3)+pET-9a-HC/B was grown in terrific broth media at 18°C without induction. Under these conditions, the yield was an order of magnitude higher than previously reported. Standard purification of the protein using a nickel column resulted in a low purity of HC/B. However, the addition of an acidic wash step prior to protein elution released a major protein contaminant and significantly increased the purity level. Mass spectrometry analysis identified the contaminant as ArnA, an E. coli protein that often contaminates recombinant His-tagged protein preparations. The purified HC/B was highly immunogenic, protecting mice from a 10(6) LD50 challenge after a single vaccination and generating a neutralizing titer of 50IU/ml after three immunizations. Moreover, the functionality of the protein was preserved, as it inhibited BoNT/B intoxication in vivo, presumably due to blockade of the neurotoxin protein receptor synaptotagmin.

Short- and long-term outcomes of pulmonary exposure to a sublethal dose of ricin in mice.

Ricin, an extremely potent toxin produced from the seeds of castor plant, Ricinus communis, is ribosome-inactivating protein that blocks cell-protein synthesis. It is considered a biological threat due to worldwide availability of castor beans, massive quantities as a by-product of castor oil production, high stability and ease of production. The consequence of exposure to lethal dose of ricin was extensively described in various animal models. However, it is assumed that in case of aerosolized ricin bioterror attack, the majority of individuals would be exposed to sublethal doses rather than to lethal ones. Therefore, the purpose of current study was to assess short- and long-term effects on physiological parameters and function following sublethal pulmonary exposure. We show that in the short-term, sublethal exposure of mice to ricin resulted in acute lung injury, including interstitial pneumonia, cytokine storm, neutrophil influx, edema and cellular death. This damage was manifested in reduced lung performance and physiological function. Interestingly, although in the long-term, mice recovered from acute lung damage and restored pulmonary and physiological functionality, the reparative process was associated with lasting fibrotic lesions. Therefore, restriction of short-term acute phase of the disease and management of long-term pulmonary fibrosis by medical countermeasures is expected to facilitate the quality of life of exposed survivors.

Single-dose methotrexate-based protocol for the treatment of caesarean scar pregnancy and successive pregnancy outcomes.

The incidence of caesarean scar pregnancy (CSP) increases in recent years. Yet, the best mode of treatment and its effects on successive pregnancies is not well established. The aim of this study was to investigate the success rate of single-dose methotrexate (MTX) in the management of CSP, and the outcomes of subsequent pregnancies in a retrospective cohort study. All women who were treated for CSPs between the years 2011 and 2019 were included. Treatment included systemic MTX and ultrasound-guided needle aspiration (UGNA) in cases with active foetal heartbeat. Overall, 34 women were diagnosed with CSP, of whom 31 were treated with systemic MTX. Twelve patients (38.7%) needed additional curettage or hysteroscopy. The only identified risk factor for failure of MTX-based treatment was time interval between the previous caesarean delivery and CSP (22 vs 34 months, p = 0.04). Twelve women had a subsequent pregnancy. Five pregnancies ended in term delivery, three in preterm delivery, three in abortion and one woman had a recurrent CSP. The study conclusion is that a single dose MTX with UGNA in cases of active heartbeat is an effective mode of treatment in cases of CSP with good sequential pregnancy outcomes. Longer time interval from the previous caesarean delivery was identified as a risk factor for failure of conservative management.

Trial of Labor After Cesarean of Small for Gestational Age Neonates Among Women with No Prior Vaginal Delivery - a Retrospective Study.

To evaluate the characteristics and outcomes of women who had never delivered vaginally and underwent a trial of labor after cesarean (TOLAC) of small for gestational age (SGA) neonates, and to identify risk factors for unplanned repeat cesarean delivery. A retrospective cohort study from two tertiary medical centers. All women undergoing a TOLAC with no prior vaginal delivery, delivering a singleton SGA neonate at term between 2005 and 2020 were included. Factors associated with successful vaginal delivery were examined by a multivariable analysis. Of the 255 women who met the inclusion criteria and underwent TOLAC, 72.2% delivered vaginally. In a multivariable analysis, maternal height [adjusted odds ratio (aOR) (95% CI): 1.10 (1.02-1.19), p = 0.012] and epidural administration [aOR (95% CI): 2.78 (1.0-7.73), p = 0.050] were positively independently associated with TOLAC success, and hypertensive disorders were negatively independently associated with TOLAC success [aOR (95% CI): 0.52 (0.004-0.74), p = 0.029]. The success rate of TOLAC among women with no prior vaginal delivery, delivering a SGA neonate is relatively high. Maternal height, hypertensive disorders, and epidural administration are independent factors associated with TOLAC success. Epidural administration is a modifiable factor and should be taken in consideration during TOLAC management.

Centaur antibodies: Engineered chimeric equine-human recombinant antibodies.

Hyper-immune antisera from large mammals, in particular horses, are routinely used for life-saving anti-intoxication intervention. While highly efficient, the use of these immunotherapeutics is complicated by possible recipient reactogenicity and limited availability. Accordingly, there is an urgent need for alternative improved next-generation immunotherapies to respond to this issue of high public health priority. Here, we document the development of previously unavailable tools for equine antibody engineering. A novel primer set, EquPD v2020, based on equine V-gene data, was designed for efficient and accurate amplification of rearranged horse antibody V-segments. The primer set served for generation of immune phage display libraries, representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins. Highly specific scFv clones were selected and expressed as full-length antibodies, carrying equine V-genes and human Gamma1/Lambda constant genes, to be referred as "Centaur antibodies". Preliminary assessment in a murine model of botulism established their therapeutic potential. The experimental approach detailed in the current report, represents a valuable tool for isolation and engineering of therapeutic equine antibodies.

High Cell Density Cultivation Process for the Expression of Botulinum Neurotoxin a Receptor Binding Domain.

The receptor-binding domain of botulinum neurotoxin (HC fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale HC fragment expression. A simple translation of the growth conditions from shake flasks to a batch fermentation process resulted in limited culture growth and protein expression (OD of 11 and volumetric protein yields of 123 mg/L). Conducting fed-batch fermentation with rich media and continuous nutrient supplementation significantly improved culture growth (OD of 40.3) and protein expression (1093 mg/L). A further increase in HC fragment yield was achieved by high cell density cultivation (HCDC). The bacterium was grown in a defined medium and with a combined bolus/continuous feed of nutrients to maintain desired oxygen levels and prevent acetate accumulation. The final OD of the process was 260, and the volumetric yield of the HC fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified HC fragments, produced by HCDC, exhibited typical biochemical and protective characteristics in mice. Taken together, the advancements achieved in this study promote large-scale production of the HC fragment in E. coli for use in anti-botulism vaccines.

Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope.

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes.

Immunologic and Protective Properties of Subunit- vs. Whole Toxoid-Derived Anti-Botulinum Equine Antitoxin.

Botulism is a paralytic disease caused by botulinum neurotoxins (BoNTs). Equine antitoxin is currently the standard therapy for botulism in human. The preparation of equine antitoxin relies on the immunization of horses with botulinum toxoid, which suffers from low yield and safety limitations. The Hc fragment of BoNTs was suggested to be a potent antibotulinum subunit vaccine. The current study presents a comparative evaluation of equine-based toxoid-derived antitoxin (TDA) and subunit-derived antitoxin (SDA). The potency of recombinant Hc/A, Hc/B, and Hc/E in mice was similar to that of toxoids of the corresponding serotypes. A single boost with Hc/E administered to a toxoid E-hyperimmune horse increased the neutralizing antibody concentration (NAC) from 250 to 850 IU/mL. Immunization of naïve horses with the recombinant subunits induced a NAC comparable to that of horses immunized with the toxoid. SDA and TDA bound common epitopes on BoNTs, as demonstrated by an in vitro competition binding assay. In vivo, SDA and TDA showed similar efficacy when administered to guinea pigs postexposure to a lethal dose of botulinum toxins. Collectively, the results of the current study suggest that recombinant BoNT subunits may replace botulinum toxoids as efficient and safe antigens for the preparation of pharmaceutical anti-botulinum equine antitoxins.

A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism.

Antitoxin, the only licensed drug therapy for botulism, neutralizes circulating botulinum neurotoxin (BoNT). However, antitoxin is no longer effective when a critical amount of BoNT has already entered its target nerve cells. The outcome is a chronic phase of botulism that is characterized by prolonged paralysis. In this stage, blocking toxin activity within cells by next-generation intraneuronal anti-botulinum drugs (INABDs) may shorten the chronic phase of the disease and accelerate recovery. However, there is a lack of adequate animal models that simulate the chronic phase of botulism for evaluating the efficacy of INABDs. Herein, we report the development of a rabbit model for the chronic phase of botulism, induced by intoxication with a sublethal dose of BoNT. Spirometry monitoring enabled us to detect deviations from normal respiration and to quantitatively define the time to symptom onset and disease duration. A 0.85 rabbit intramuscular median lethal dose of BoNT/A elicited the most consistent and prolonged disease duration (mean = 11.8 days, relative standard deviation = 27.9%) that still enabled spontaneous recovery. Post-exposure treatment with antitoxin at various time points significantly shortened the disease duration, providing a proof of concept that the new model is adequate for evaluating novel therapeutics for botulism.

A Rabbit Model for Prolonged Continuous Intravenous Infusion Via a Peripherally Inserted Central Catheter.

Medical treatment may require the continuous intravenous (IV) infusion of drugs to sustain the therapeutic blood concentration and to minimize dosing errors. Animal disease models that ultimately mimic the intended use of new potential drugs via a continuous IV infusion in unrestrained, free roaming animals are required. While peripherally inserted central catheters (PICCs) and other central line techniques for prolonged IV infusion of drugs are prevalent in the clinic, continuous IV infusion methods in an animal model are challenging and limited. In most cases, continuous IV infusion methods require surgical knowledge as well as expensive and complicated equipment. In the current work, we established a novel rabbit model for prolonged continuous IV infusion by inserting a PICC line from the marginal ear vein to the superior vena cava and connecting it to an externally carried ambulatory infusion pump. Either saline or a clinically relevant formulation could be steadily and continuously infused at 3-6 ml/h for 11 consecutive days into freely moving rabbits while maintaining normal body temperature, weight, and respiration physiology, as determined by daily spirometry. This new model is simple to execute and can advance the ability to administer and test new drug candidates.

Crystal structure of an inverting GH 43 1,5-alpha-L-arabinanase from Geobacillus stearothermophilus complexed with its substrate.

Arabinanases are glycosidases that hydrolyse alpha-(1-->5)- arabinofuranosidic linkages found in the backbone of the pectic polysaccharide arabinan. Here we describe the biochemical characterization and the enzyme-substrate crystal structure of an inverting family 43 arabinanase from Geobacillus stearothermophilus T-6 (AbnB). Based on viscosity and reducing power measurements, and based on product analysis for the hydrolysis of linear arabinan by AbnB, the enzyme works in an endo mode of action. Isothermal titration calorimetry studies of a catalytic mutant with various arabino-oligosaccharides suggested that the enzyme active site can accommodate at least five arabinose units. The crystal structure of AbnB was determined at 1.06 A (1 A=0.1 nm) resolution, revealing a single five-bladed-beta-propeller fold domain. Co-crystallization of catalytic mutants of the enzyme with different substrates allowed us to obtain complex structures of AbnBE201A with arabinotriose and AbnBD147A with arabinobiose. Based on the crystal structures of AbnB together with its substrates, the position of the three catalytic carboxylates: Asp27, the general base; Glu201, the general acid; and Asp147, the pKa modulator, is in agreement with their putative catalytic roles. In the complex structure of AbnBE201A with arabinotriose, a single water molecule is located 2.8 A from Asp27 and 3.7 A from the anomeric carbon. The position of this water molecule is kept via hydrogen bonding with a conserved tyrosine (Tyr229) that is 2.6 A distant from it. The location of this molecule suggests that it can function as the catalytic water molecule in the hydrolysis reaction, resulting in the inversion of the anomeric configuration of the product.