Partitioning and extraction of collagenase from Penicillium aurantiogriseum in poly(ethylene glycol)/phosphate aqueous two-phase system

Purification of collagenase produced by Penicillium aurantiogriseum URM4622 was carried using a PEG/phosphate aqueous two-phase system (ATPS). A 2(3)-full experimental design was used to investigate the influence of PEG molar mass, PEG concentration and phosphate concentration on the selected responses, namely partition coefficient, activity yield and purification factor. The ATPS was composed of PEG (molar mass of 550, 1500 and 4000 g/mol) at concentrations of 15.0, 17.5 and 20.0% (w/w) and phosphate at concentrations of 12.5, 15.0 and 17.5% (w/w). The best results of one-step extraction of collagenase from the fermentation broth (partition coefficient of 1.01, activity yield of 242% and purification factor of 23.5) were obtained at pH 6.0 using 20.0% (w/w) PEG 550 and 17.5% (w/w) phosphate. The results of this preliminary study demonstrate that the selected ATPS is satisfactorily selective for the extraction of such a collagenase. (C) 2012 Elsevier B.V. All rights reserved.

A variational approach to 3D geometry reconstruction from two or multiple views

[EN] In the last years we have developed some methods for 3D reconstruction. First we began with the problem of reconstructing a 3D scene from a stereoscopic pair of images. We developed some methods based on energy functionals which produce dense disparity maps by preserving discontinuities from image boundaries. Then we passed to the problem of reconstructing a 3D scene from multiple views (more than 2). The method for multiple view reconstruction relies on the method for stereoscopic reconstruction. For every pair of consecutive images we estimate a disparity map and then we apply a robust method that searches for good correspondences through the sequence of images. Recently we have proposed several methods for 3D surface regularization. This is a postprocessing step necessary for smoothing the final surface, which could be afected by noise or mismatch correspondences. These regularization methods are interesting because they use the information from the reconstructing process and not only from the 3D surface. We have tackled all these problems from an energy minimization approach. We investigate the associated Euler-Lagrange equation of the energy functional, and we approach the solution of the underlying partial differential equation (PDE) using a gradient descent method.

Comparative phylogeography of two co-distributed arctic-alpine freshwater insect species in Europe

The distribution pattern of European arctic-alpine disjunct species is of growing interest among biogeographers due to the arising variety of inferred demographic histories. In this thesis I used the co-distributed mayfly Ameletus inopinatus and the stonefly Arcynopteryx compacta as model species to investigate the European Pleistocene and Holocene history of stream-inhabiting arctic-alpine aquatic insects. I used last glacial maximum (LGM) species distribution models (SDM) to derive hypotheses on the glacial survival during the LGM and the recolonization of Fennoscandia: 1) both species potentially survived glacial cycles in periglacial, extra Mediterranean refugia, and 2) postglacial recolonization of Fennoscandia originated from these refugia. I tested these hypotheses using mitochondrial sequence (mtCOI) and species specific microsatellite data. Additionally, I used future SDM to predict the impact of climate change induced range shifts and habitat loss on the overall genetic diversity of the endangered mayfly A. inopinatus.rnI observed old lineages, deep splits, and almost complete lineage sorting of mtCOI sequences between mountain ranges. These results support the hypothesis that both species persisted in multiple periglacial extra-Mediterranean refugia in Central Europe during the LGM. However, the recolonization of Fennoscandia was very different between the two study species. For the mayfly A. inopinatus I found strong differentiation between the Fennoscandian and all other populations in sequence and microsatellite data, indicating that Fennoscandia was recolonized from an extra European refugium. High mtCOI genetic structure within Fennoscandia supports a recolonization of multiple lineages from independent refugia. However, this structure was not apparent in the microsatellite data, consistent with secondary contact without sexual incompability. In contrast, the stonefly A. compacta exhibited low genetic structure and shared mtCOI haplotypes among Fennoscandia and the Black Forest, suggesting a shared Pleistocene refugium in the periglacial tundrabelt. Again, there is incongruence with the microsatellite data, which could be explained with ancestral polymorphism or female-biased dispersal. Future SDM projects major regional habitat loss for the mayfly A. inopinatus, particularly in Central European mountain ranges. By relating these range shifts to my population genetic results, I identified conservation units primarily in Eastern Europe, that if preserved would maintain high levels of the present-day genetic diversity of A. inopinatus and continue to provide long-term suitable habitat under future climate warming scenarios.rnIn this thesis I show that despite similar present day distributions the underlying demographic histories of the study species are vastly different, which might be due to differing dispersal capabilities and niche plasticity. I present genetic, climatic, and ecological data that can be used to prioritize conservation efforts for cold-adapted freshwater insects in light of future climate change. Overall, this thesis provides a next step in filling the knowledge gap regarding molecular studies of the arctic-alpine invertebrate fauna. However, there is continued need to explore the phenomenon of arctic-alpine disjunctions to help understand the processes of range expansion, regression, and lineage diversification in Europe’s high latitude and high altitude biota.

Deposizione elettrochimica di esacianoferrato di rame tramite un metodo "a due step"; caratterizzazione e valutazione delle prestazioni

In this thesis we have identified two electrochemical procedures for preparing two compounds of copper hexacyanoferrate (CuHCF) films with different compositions and structures. The deposition were carried out using a “two steps” method consisting in electrochemical oxidation of previously deposited metallic copper on carbon substrates (glassy carbon and graphite foil electrodes) in K3[Fe(CN)6] solution. Both films, CuHCF-methodA and CuHCF-methodB, were characterized by cyclic voltammetry (GC) and their study using XANES spectroscopy revealed evidence of different structures. Additionally, insertion and extraction of different cations (Na+, K+, Mg2+, Al3+ and Cs+) were performed and the results indicate that CuHCF-methodA has slightly better performances and operational stability than CuHCF-methodB. Data from galvanostatic charge-discharge tests confirme the latter observation. An application for amperometric detection of H2O2 and SEM micrographs are also reported for both films (method A and B). Comparing these results with a previous work of our research group, seems that the deposition of two different compounds using methodA and methodB is due to the different stoichiometry of ions Cu2+ e [Fe(CN)6]3– created near electrode surface during the dissolution step.

Two ways to common knowledge

It is not clear what a system for evidence-based common knowledge should look like if common knowledge is treated as a greatest fixed point. This paper is a preliminary step towards such a system. We argue that the standard induction rule is not well suited to axiomatize evidence-based common knowledge. As an alternative, we study two different deductive systems for the logic of common knowledge. The first system makes use of an induction axiom whereas the second one is based on co-inductive proof theory. We show the soundness and completeness for both systems.

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure--in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.

Gadolinium diethylenetriaminepentaacetate enhancement kinetics in the menisci of asymptomatic subjects: a first step towards a dedicated dGEMRIC (delayed gadolinium-enhanced MRI of cartilage)-like protocol for biochemical imaging of the menisci

It was our aim to investigate the gadolinium diethylenetriaminepentaacetate (Gd-DTPA(2-) ) enhancement kinetics in the menisci of the knee joint over a prolonged period of time. Six asymptomatic volunteers (four men and two women; mean age, 25 ± 2.4 years) were enrolled. Sagittal, T(1) -weighted, spin-echo MR sequences of the right knee joint were obtained at 3 T. Imaging was performed before (baseline), 1 h after and in half-hour intervals up to 9 h after the intravenous administration of 0.2 mmol/kg of Gd-DTPA(2-) . To measure the rates of contrast enhancement relative to the baseline, regions of interest that covered the anterior and posterior horns of the medial and lateral meniscus were defined on each of two adjacent sections, and enhancement curves were constructed. An enhancement peak between 2.5 and 4.5 h after Gd-DTPA(2-) administration was observed, and analysis of variance also revealed no significant difference (p=0.94), in terms of enhancement, within this time interval. Pair-wise, post hoc testing also revealed no significant differences between 2.5 and 3, 3 and 3.5, 3.5 and 4, and 4 and 4.5 h post Gd-DTPA(2-) application. Our preliminary data therefore suggest that the time window suitable for a dGEMRIC (delayed gadolinium-enhanced MRI of cartilage)-like T(1) mapping of the menisci is relatively short, and lies between 2.5 and 4.5 h after Gd-DTPA(2-) injection.

The use of a decremental dose regimen in patients treated with a chronic low-dose step-up protocol for WHO Group II anovulation: a prospective randomized multicentre study

BACKGROUND: In women with chronic anovulation, the choice of the FSH starting dose and the modality of subsequent dose adjustments are critical in controlling the risk of overstimulation. The aim of this prospective randomized study was to assess the efficacy and safety of a decremental FSH dose regimen applied once the leading follicle was 10-13 mm in diameter in women treated for WHO Group II anovulation according to a chronic low-dose (CLD; 75 IU FSH for 14 days with 37.5 IU increment) step-up protocol. METHODS: Two hundred and nine subfertile women were treated with recombinant human FSH (r-hFSH) (Gonal-f) for ovulation induction according to a CLD step-up regimen. When the leading follicle reached a diameter of 10-13 mm, 158 participants were randomized by means of a computer-generated list to receive either the same FSH dose required to achieve the threshold for follicular development (CLD regimen) or half of this FSH dose [sequential (SQ) regimen]. HCG was administered only if not more than three follicles >or=16 mm in diameter were present and/or serum estradiol (E(2)) values were <1200 pg/ml. The primary outcome measure was the number of follicles >or=16 mm in size at the time of hCG administration. RESULTS: Clinical characteristics and ovarian parameters at the time of randomization were similar in the two groups. Both CLD and SQ protocols achieved similar follicular growth as regards the total number of follicles and medium-sized or mature follicles (>/=16 mm: 1.5 +/- 0.9 versus 1.4 +/- 0.7, respectively). Furthermore, serum E(2) levels were equivalent in the two groups at the time of hCG administration (441 +/- 360 versus 425 +/- 480 pg/ml for CLD and SQ protocols, respectively). The rate of mono-follicular development was identical as well as the percentage of patients who ovulated and achieved pregnancy. CONCLUSIONS: The results show that the CLD step-up regimen for FSH administration is efficacious and safe for promoting mono-follicular ovulation in women with WHO Group II anovulation. This study confirms that maintaining the same FSH starting dose for 14 days before increasing the dose in step-up regimen is critical to adequately control the risk of over-response. Strict application of CLD regimen should be recommended in women with WHO Group II anovulation.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

The influence of zero-value subtraction on the performance of two laser fluorescence devices for detecting occlusal caries in vitro

BACKGROUND: The aim of this study was to evaluate the influence of zero-value subtraction on the performance of two laser fluorescence (LF) devices developed to detect occlusal caries. METHODS: The authors selected 119 permanent molars. Two examiners assessed three areas (cuspal, middle and cervical) of both mesial and distal portions of the buccal surface and one occlusal site using an LF device and an LF pen. For each tooth, the authors subtracted the value measured in the cuspal, middle and cervical areas in the buccal surface from the value measured in the respective occlusal site. RESULTS: The authors observed differences among the readings for both devices in the cuspal, middle and cervical areas in the buccal surface as well as differences for both devices with and without the zero-value subtraction in the occlusal surface. When the authors did not perform the zero-value subtraction, they found statistically significant differences for sensitivity and accuracy for the LF device. When this was done with the LF pen, specificity increased and sensitivity decreased significantly. CONCLUSIONS: For the LF device, the zero-value subtraction decreased the sensitivity. For this reason, the authors concluded that clinicians can obtain measures with the LF device effectively without using zero-value subtraction. For the LF pen, however, the absence of the zero-value subtraction changed both the sensitivity and specificity, and so the authors concluded that clinicians should not eliminate this step from the procedure. CLINICAL IMPLICATIONS: When using the LF device, clinicians might not need to perform the zero-value subtraction; however, for the LF pen, clinicians should do so.

Risk prediction of fever in neutropenia in children with cancer: a step towards individually tailored supportive therapy?

BACKGROUND: Fever in severe chemotherapy-induced neutropenia (FN) is the most frequent manifestation of a potentially lethal complication of current intensive chemotherapy regimens. This study aimed at establishing models predicting the risk of FN, and of FN with bacteremia, in pediatric cancer patients. METHODS: In a single-centre cohort study, characteristics potentially associated with FN and episodes of FN were retrospectively extracted from charts. Poisson regression accounting for chemotherapy exposure time was used for analysis. Prediction models were constructed based on a derivation set of two thirds of observations, and validated based on the remaining third of observations. RESULTS: In 360 pediatric cancer patients diagnosed and treated for a cumulative chemotherapy exposure time of 424 years, 629 FN were recorded (1.48 FN per patient per year, 95% confidence interval (CI), 1.37-1.61), 145 of them with bacteremia (23% of FN; 0.34; 0.29-0.40). More intensive chemotherapy, shorter time since diagnosis, bone marrow involvement, central venous access device (CVAD), and prior FN were significantly and independently associated with a higher risk to develop both FN and FN with bacteremia. The prediction models explained more than 30% of the respective risks. CONCLUSIONS: The two models predicting FN and FN with bacteremia were based on five easily accessible clinical variables. Before clinical application, they need to be validated by prospective studies.

"Rapid two-stage" Norwood operation in a child with multiorgan failure

The Norwood I operation continues to be a procedure with significant operative mortality. One well-accepted risk factor for death after the first step of the Norwood operation is critical preoperative status. We describe herein a new concept for the treatment of patients with hypoplastic left heart syndrome (HLHS) in very poor preoperative condition. This is a case report of a child who was born in a rural hospital. On the second day of life he was referred to our center in multiorgan failure. There were signs of liver dysfunction and the child was anuric. Therapy was started immediately with prostaglandin and vasodilators as well as diuretics, milrinone, and dobutamine. However, systemic perfusion continued to be insufficient. Finally, the child was placed on a ventilator. On the fourth day of life, bilateral pulmonary artery (PA) banding was performed and circulation stabilized immediately. Two hours after the operation urine output started. Liver function stabilized over the next couple of days. Two days after PA banding the child was weaned from the ventilator. On the 12th day of life a Norwood operation with PA debanding and a right ventricle-PA conduit was performed, and 2 days postoperatively the child was weaned from the ventilator. Twenty days after the operation he was discharged home. When the boy was 4 months old a bidirectional cavopulmonary anastomosis was performed. In selected cases of patients with HLHS with very poor hemodynamic conditions, a rapid two-stage approach with bilateral banding followed by a Norwood operation after cardiac stabilization can be recommended.

Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway

Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.

ANKLE IMPEDANCE AND ANKLE ANGLES DURING STEP TURN AND STRAIGHT WALK: IMPLICATIONS FOR THE DESIGN OF A STEERABLE ANKLE-FOOT PROSTHETIC ROBOT

During locomotion, turning is a common and recurring event which is largely neglected in the current state-of-the-art ankle-foot prostheses, forcing amputees to use different steering mechanisms for turning, compared to non-amputees. A better understanding of the complexities surrounding lower limb prostheses will lead to increased health and well-being of amputees. The aim of this research is to develop a steerable ankle-foot prosthesis that mimics the human ankle mechanical properties. Experiments were developed to estimate the mechanical impedance of the ankle and the ankles angles during straight walk and step turn. Next, this information was used in the design of a prototype, powered steerable ankle-foot prosthesis with two controllable degrees of freedom. One of the possible approaches in design of the prosthetic robots is to use the human joints’ parameters, especially their impedance. A series of experiments were conducted to estimate the stochastic mechanical impedance of the human ankle when muscles were fully relaxed and co-contracting antagonistically. A rehabilitation robot for the ankle, Anklebot, was employed to provide torque perturbations to the ankle. The experiments were performed in two different configurations, one with relaxed muscles, and one with 10% of maximum voluntary contraction (MVC). Surface electromyography (sEMG) was used to monitor muscle activation levels and these sEMG signals were displayed to subjects who attempted to maintain them constant. Time histories of ankle torques and angles in the lateral/medial (LM) directions, inversion-eversion (IE), and dorsiflexionplantarflexion (DP) were recorded. Linear time-invariant transfer functions between the measured torques and angles were estimated providing an estimate of ankle mechanical impedance. High coherence was observed over a frequency range up to 30 Hz. The main effect of muscle activation was to increase the magnitude of ankle mechanical impedance in all degrees of freedom of the ankle. Another experiment compared the three-dimensional angles of the ankle during step turn and straight walking. These angles were measured to be used for developing the control strategy of the ankle-foot prosthesis. An infrared camera system was used to track the trajectories and angles of the foot and leg. The combined phases of heel strike and loading response, mid stance, and terminal stance and pre-swing were determined and used to measure the average angles at each combined phase. The Range of motion (ROM) in IE increased during turning while ML rotation decreased and DP changed the least. During the turning step, ankle displacement in DP started with similar angles to straight walk and progressively showed less plantarflexion. In IE, the ankle showed increased inversion leaning the body toward the inside of the turn. ML rotation initiated with an increased medial rotation during the step turn relative to the straight walk transitioning to increased lateral rotation at the toe off. A prototype ankle-foot prosthesis capable of controlling both DP and IE using a cable driven mechanism was developed and assessed as part of a feasibility study. The design is capable of reproducing the angles required for straight walk and step turn; generates 712N of lifting force in plantarflexion, and shows passive stiffness comparable to a nonload bearing ankle impedance. To evaluate the performance of the ankle-foot prosthesis, a circular treadmill was developed to mimic human gait during steering. Preliminary results show that the device can appropriately simulate human gait with loading and unloading the ankle joint during the gait in circular paths.

Separation of Cadmium from Zinc in the Purification Step of the Leaching Cycle

The purpose of this investigation is to determine the effect of the factors listed previously by conducting a series of tests that will indicate the ex­tent to which the purification is influenced by time, temperature, zinc oust size, zinc dust quantity, iron concentration, two stage precipitation, and aeration.