bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A
Authors: de Azevedo Marcela , Karczewski Jurgen , Lefévre François , Azevedo Vasco , Miyoshi Anderson , Wells Jerry M , Langella Philippe , Chatel Jean-Marc 
Published: December 2012

Abstract

Background
The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant invasive LL expressing Fibronectin Binding Protein A of Staphylococcus aureus (LL-FnBPA+) or Internalin A of Listeria monocytogenes (LL-InlA+), were able to deliver and trigger DNA expression by epithelial cells, either in vitro or in vivo. InlA does not bind to its receptor, the murine E-cadherin, thus limiting the use of LL-InlA+ in in vivo murine models. Moreover, FnBPA binds to its receptors, integrins, via fibronectin introducing another limiting factor. In order to avoid the limitations of LL-InlA+ and LL-FnBPA+, a new L. lactis strain was engineered to produce a previously described mutated form of InlA (LL-mInlA+) allowing the binding of mInlA on murine E-cadherin.

Results
After showing the expression of mInLA at the surface of LL-mInlA+ strain, in vitro gentamycin survival assay in Caco-2 cells showed that LL-mInlA+ is 1000 times more invasive than LL. LL-mInlA+ invasivity was also validated by fluorescence microscopy. LL and LL-mInlA+ were transformed with pValacBLG, a plasmid containing the cDNA of bovine β-Lactoglobulin (BLG), resulting in strains LL-BLG and LL-mInlA+BLG. The plasmid transfer in vitro using LL-mInlA+BLG was increased 10 times compared to LL-BLG. Moreover, the number of mice producing BLG in isolated enterocytes after oral administration of LL-mInlA+BLG in vivo was slightly higher than after oral administration of LL-BLG.

Conclusions
We confirmed in this study that the production of mInlA at the surface of L. lactis is a promising strategy for plasmid transfer in vitro and in vivo.
Determinants of acceptance of cervical cancer screening in Dar es Salaam, Tanzania
Authors: Kahesa Crispin , Kjaer Susanne , Mwaiselage Julius , Ngoma Twalib , Tersbol Britt , Dartell Myassa , Rasch Vibeke 
Published: December 2012

Abstract

Objective
To describe how demographic characteristics and knowledge of cervical cancer influence screening acceptance among women living in Dar es Salaam, Tanzania.

Methods
Multistage cluster sampling was carried out in 45 randomly selected streets in Dar es Salaam. Women between the ages of 25–59 who lived in the sampled streets were invited to a cervical cancer screening; 804 women accepted and 313 rejected the invitation. Information on demographic characteristics and knowledge of cervical cancer were obtained through structured questionnaire interviews.

Results
Women aged 35–44 and women aged 45–59 had increased ORs of 3.52 and 7.09, respectively, for accepting screening. Increased accepting rates were also found among single women (OR 2.43) and among women who had attended primary or secondary school (ORs of 1.81 and 1.94). Women who had 0–2 children were also more prone to accept screening in comparison with women who had five or more children (OR 3.21). Finally, knowledge of cervical cancer and awareness of the existing screening program were also associated with increased acceptance rates (ORs of 5.90 and 4.20).

Conclusion
There are identifiable subgroups where cervical cancer screening can be increased in Dar es Salaam. Special attention should be paid to women of low education and women of high parity. In addition, knowledge and awareness raising campaigns that goes hand in hand with culturally acceptable screening services will likely lead to an increased uptake of cervical cancer screening.
Multidisciplinary approach to management of maternal asthma (MAMMA [copyright]): the PROTOCOL for a randomized controlled trial
Authors: Lim Angelina , Stewart Kay , Abramson Michael J , Walker Susan P , George Johnson 
Published: December 2012

Abstract

Background
Uncontrolled asthma during pregnancy is associated with the maternal hazards of disease exacerbation, and perinatal hazards including intrauterine growth restriction and preterm birth. Interventions directed at achieving better asthma control during pregnancy should be considered a high priority in order to optimise both maternal and perinatal outcomes. Poor compliance with prescribed asthma medications during pregnancy and suboptimal prescribing patterns to pregnant women have both been shown to be contributing factors that jeopardise asthma control. The aim is to design and evaluate an intervention involving multidisciplinary care for women experiencing asthma in pregnancy.

Methods/design
A pilot single-blinded parallel-group randomized controlled trial testing a Multidisciplinary Approach to Management of Maternal Asthma (MAMMA©) which involves education and regular monitoring. Pregnant women with asthma will be recruited from antenatal clinics in Victoria, Australia. Recruited participants, stratified by disease severity, will be allocated to the intervention or the usual care group in a 1:1 ratio. Both groups will be followed prospectively throughout pregnancy and outcomes will be compared between groups at three and six months after recruitment to evaluate the effectiveness of this intervention. Outcome measures include Asthma Control Questionnaire (ACQ) scores, oral corticosteroid use, asthma exacerbations and asthma related hospital admissions, and days off work, preventer to reliever ratio, along with pregnancy and neonatal adverse events at delivery. The use of FEV1/FEV6 will be also investigated during this trial as a marker for asthma control.

Discussion
If successful, this model of care could be widely implemented in clinical practice and justify more funding for support services and resources for these women. This intervention will also promote awareness of the risks of poorly controlled asthma and the need for a collaborative, multidisciplinary approach to asthma management during pregnancy. This is also the first study to investigate the use of FEV1/FEV6 as a marker for asthma control during pregnancy.

Trial registration
Australian New Zealand Clinical Trials Registry (ACTRN12612000681853)
Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha
Authors: Moussa Manal , Ibrahim Mahmoud , El Ghazaly Maria , Rohde Jan , Gnoth Stefan , Anton Andreas , Kensy Frank , Mueller Frank 
Published: December 2012

Abstract

Background
Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system.

Results
The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved.

Conclusion
The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.
VEGF induces sensory and motor peripheral plasticity, alters bladder function, and promotes visceral sensitivity
Authors: Malykhina Anna P , Lei Qi , Erickson Chris S , Epstein Miles L , Saban Marcia R , Davis Carole A , Saban Ricardo 
Published: December 2012

Abstract

Background
This work tests the hypothesis that bladder instillation with vascular endothelial growth factor (VEGF) modulates sensory and motor nerve plasticity, and, consequently, bladder function and visceral sensitivity.
In addition to C57BL/6J, ChAT-cre mice were used for visualization of bladder cholinergic nerves. The direct effect of VEGF on the density of sensory nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) and cholinergic nerves (ChAT) was studied one week after one or two intravesical instillations of the growth factor.
To study the effects of VEGF on bladder function, mice were intravesically instilled with VEGF and urodynamic evaluation was assessed. VEGF-induced alteration in bladder dorsal root ganglion (DRG) neurons was performed on retrogradly labeled urinary bladder afferents by patch-clamp recording of voltage gated Na+ currents. Determination of VEGF-induced changes in sensitivity to abdominal mechanostimulation was performed by application of von Frey filaments.

Results
In addition to an overwhelming increase in TRPV1 immunoreactivity, VEGF instillation resulted in an increase in ChAT-directed expression of a fluorescent protein in several layers of the urinary bladder. Intravesical VEGF caused a profound change in the function of the urinary bladder: acute VEGF (1 week post VEGF treatment) reduced micturition pressure and longer treatment (2 weeks post-VEGF instillation) caused a substantial reduction in inter-micturition interval. In addition, intravesical VEGF resulted in an up-regulation of voltage gated Na+ channels (VGSC) in bladder DRG neurons and enhanced abdominal sensitivity to mechanical stimulation.

Conclusions
For the first time, evidence is presented indicating that VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. The VEGF pathway is being proposed as a key modulator of neural plasticity in the pelvis and enhanced VEGF content may be associated with visceral hyperalgesia, abdominal discomfort, and/or pelvic pain.
The biochemistry of acetaminophen hepatotoxicity and rescue: a mathematical model
Authors: Ben-Shachar Rotem , Chen Yifei , Luo Shishi , Hartman Catherine , Reed Michael , Nijhout H Frederik 
Published: December 2012

Abstract

Background
Acetaminophen (N-acetyl-para-aminophenol) is the most widely used over-the-counter or prescription painkiller in the world. Acetaminophen is metabolized in the liver where a toxic byproduct is produced that can be removed by conjugation with glutathione. Acetaminophen overdoses, either accidental or intentional, are the leading cause of acute liver failure in the United States, accounting for 56,000 emergency room visits per year. The standard treatment for overdose is N-acetyl-cysteine (NAC), which is given to stimulate the production of glutathione.

Methods
We have created a mathematical model for acetaminophen transport and metabolism including the following compartments: gut, plasma, liver, tissue, urine. In the liver compartment the metabolism of acetaminophen includes sulfation, glucoronidation, conjugation with glutathione, production of the toxic metabolite, and liver damage, taking biochemical parameters from the literature whenever possible. This model is then connected to a previously constructed model of glutathione metabolism.

Results
We show that our model accurately reproduces published clinical and experimental data on the dose-dependent time course of acetaminophen in the plasma, the accumulation of acetaminophen and its metabolites in the urine, and the depletion of glutathione caused by conjugation with the toxic product. We use the model to study the extent of liver damage caused by overdoses or by chronic use of therapeutic doses, and the effects of polymorphisms in glucoronidation enzymes. We use the model to study the depletion of glutathione and the effect of the size and timing of N-acetyl-cysteine doses given as an antidote. Our model accurately predicts patient death or recovery depending on size of APAP overdose and time of treatment.

Conclusions
The mathematical model provides a new tool for studying the effects of various doses of acetaminophen on the liver metabolism of acetaminophen and glutathione. It can be used to study how the metabolism of acetaminophen depends on the expression level of liver enzymes. Finally, it can be used to predict patient metabolic and physiological responses to APAP doses and different NAC dosing strategies.
Prognostic significance of thyroid or cricoid cartilage invasion in laryngeal or hypopharyngeal cancer treated with organ preserving strategies
Authors: Wagner Marcus M , Curé Joel K , Caudell Jimmy J , Spencer Sharon A , Nabell Lisle M , Carroll William R , Bonner James A 
Published: December 2012

Abstract

Background
The utility of definitive radiotherapy (RT) for locoregionally advanced squamous cell carcinoma (SCC) of the larynx or hypopharynx in the setting of thyroid or cricoid cartilage invasion (TCCI) is controversial. A retrospective review of our experience was performed.

Methods
Our institutional database of patients with SCC of the head and neck treated with radiotherapy (90% received concurrent systemic therapy) between 1995 and 2009 was queried. We identified 87 patients with T3-4 laryngeal or T4 hypopharyngeal cancer for whom initial head and neck imaging was available for review. Imaging of all patients was reviewed by a single radiologist specializing in neuroradiology. The presence and extent of TCCI was determined and used for stratification.

Results
Median follow-up was 34 months. TCCI was found in 25 (29%) patients, eight limited to the inner cortex and another 17 involving both cortices. Local control (LC) was not significantly affected by TCCI limited to the inner cortex. However, TCCI involving both cortices was correlated with diminished LC at 2 years compared to the group of patients with no or minor invasion (55% vs. 81%, p=0.045). However, TCCI involving both cortices was not associated with significantly reduced rates of survival with a functional larynx, or overall survival (OS).

Conclusions
Our results suggest that the rate of LC of T3-4 laryngeal or T4 hypopharyngeal SCC treated with definitive RT is not affected by TCCI of the inner cortex. Although decreased LC was significantly associated with TCCI involving both cortices, this factor did not appear to result in reduced rates of survival with a functional larynx or OS. Therefore, organ preservation may remain an option in these patients.
Umbilical cord blood-derived mesenchymal stem cells consist of a unique population of progenitors co-expressing mesenchymal stem cell and neuronal markers capable of instantaneous neuronal differentiation
Authors: Divya Mundackal S , Roshin George E , Divya Thulasi S , Rasheed Vazhanthodi Abdul , Santhoshkumar Thankayyan R , Elizabeth Kandathil E , James Jackson , Pillai Radhakrishna M 
Published: December 2012

Abstract
Introduction
Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are self-renewing multipotent progenitors with the potential to differentiate into multiple lineages of mesoderm, in addition to generating ectodermal and endodermal lineages by crossing the germline barrier. In the present study we have investigated the ability of UCB-MSCs to generate neurons, since we were able to observe varying degrees of neuronal differentiation from a few batches of UCB-MSCs with very simple neuronal induction protocols whereas other batches required extensive exposure to combination of growth factors in a stepwise protocol. Our hypothesis was therefore that the human UCB-MSCs would contain multiple types of progenitors with varying neurogenic potential and that the ratio of the progenitors with high and low neurogenic potentials varies in different batches of UCB.

Methods
In total we collected 45 UCB samples, nine of which generated MSCs that were further expanded and characterized using immunofluorescence, fluorescence-activated cell sorting and RT-PCR analysis. The neuronal differentiation potential of the UCB-MSCs was analyzed with exposure to combination of growth factors.

Results
We could identify two different populations of progenitors within the UCB-MSCs. One population represented progenitors with innate neurogenic potential that initially express pluripotent stem cell markers such as Oct4, Nanog, Sox2, ABCG2 and neuro-ectodermal marker nestin and are capable of expanding and differentiating into neurons with exposure to simple neuronal induction conditions. The remaining population of cells, typically expressing MSC markers, requires extensive exposure to a combination of growth factors to transdifferentiate into neurons. Interesting to note was that both of these cell populations were positive for CD29 and CD105, indicating their MSC lineage, but showed prominent difference in their neurogenic potential.

Conclusion
Our results suggest that the expanded UCB-derived MSCs harbor a small unique population of cells that express pluripotent stem cell markers along with MSC markers and possess an inherent neurogenic potential. These pluripotent progenitors later generate cells expressing neural progenitor markers and are responsible for the instantaneous neuronal differentiation; the ratio of these pluripotent marker expressing cells in a batch determines the innate neurogenic potential.
Environmental interaction influences muscle activation strategy during sand-swimming in the sandfish lizard Scincus scincus
Authors: Sharpe Sarah S. , Ding Yang , Goldman Daniel I. 
Published: December 2012

SUMMARY
Animals like the sandfish lizard (Scincus scincus) that live in desert sand locomote on and within a granular medium whose resistance to intrusion is dominated by frictional forces. Recent kinematic studies revealed that the sandfish utilizes a wave of body undulation during swimming. Models predict that a particular combination of wave amplitude and wavelength yields maximum speed for a given frequency, and experiments have suggested that the sandfish targets this kinematic waveform. To investigate the neuromechanical strategy of the sandfish during walking, burial and swimming, here we use high-speed X-ray and visible light imaging with synchronized electromyogram (EMG) recordings of epaxial muscle activity. While moving on the surface, body undulation was not observed and EMG showed no muscle activation. During subsurface sand-swimming, EMG revealed an anterior-to-posterior traveling wave of muscle activation which traveled faster than the kinematic wave. Muscle activation intensity increased as the animal swam deeper into the material but was insensitive to undulation frequency. These findings were in accord with empirical force measurements, which showed that resistance force increased with depth but was independent of speed. The change in EMG intensity with depth indicates that the sandfish targets a kinematic waveform (a template) that models predict maximizes swimming speed and minimizes the mechanical cost of transport as the animal descends into granular media. The differences in the EMG pattern compared with EMG of undulatory swimmers in fluids can be attributed to the friction-dominated intrusion forces of granular media.
Real-time measurement of metabolic rate during freezing and thawing of the wood frog,Rana sylvatica: implications for overwinter energy use
Authors: Sinclair Brent J. , Stinziano Joseph R. , Williams Caroline M. , MacMillan Heath A. , Marshall Katie E. , Storey Kenneth B. 
Published: December 2012

SUMMARYEctotherms overwintering in temperate ecosystems must survive low temperatures while conserving energy to fuel post-winter reproduction. Freeze-tolerant wood frogs, Rana sylvatica, have an active response to the initiation of ice formation that includes mobilising glucose from glycogen and circulating it around the body to act as a cryoprotectant. We used flow-through respirometry to measure CO2 production () in real time during cooling, freezing and thawing. CO2 production increases sharply at three points during freeze–thaw: at +1°C during cooling prior to ice formation (total of 104±17 μl CO2 frog−1 event−1), at the initiation of freezing (565±85 μl CO2 frog−1 freezing event−1) and after the frog has thawed (564±75 μ l CO2 frog−1 freezing event−1). We interpret these increases in metabolic rate to represent the energetic costs of preparation for freezing, the response to freezing and the re-establishment of homeostasis and repair of damage after thawing, respectively. We assumed that frogs metabolise lipid when unfrozen and that carbohydrate fuels metabolism during cooling, freezing and thawing, and when frozen. We then used microclimate temperature data to predict overwinter energetics of wood frogs. Based on the freezing and melting points we measured, frogs in the field were predicted to experience as many as 23 freeze–thaw cycles in the winter of our microclimate recordings. Overwinter carbohydrate consumption appears to be driven by the frequency of freeze–thaw events, and changes in overwinter climate that affect the frequency of freeze–thaw will influence carbohydrate consumption, but changes that affect mean temperatures and the frequency of winter warm spells will modify lipid consumption.