Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/ cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological efect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specifc hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purifed A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafl inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth defciencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. Conclusions: To our knowledge, this is the frst time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specifc PDE inhibitors for therapeutic applications in many pathologies

On the Use of Fuzzy and Permutation Entropy in Hand Gesture Characterization from EMG Signals: Parameters Selection and Comparison

The surface electromyography signal (sEMG) is widely used for gesture characterization; its reliability is strongly connected to the features extracted from sEMG recordings. This study aimed to investigate the use of two complexity measures, i.e., fuzzy entropy (FEn) and permutation entropy (PEn) for hand gesture characterization. Fourteen upper limb movements, sorted into three sets, were collected on ten subjects and the performances of FEn and PEn for gesture descriptions were analyzed for different computational parameters. FEn and PEn were able to properly cluster the expected numbers of gestures, but computational parameters were crucial for ensuring clusters' separability and proper gesture characterization. FEn and PEn were also compared with other eighteen classical time and frequency domain features through the minimum redundancy maximum relevance algorithm and showed the best predictive importance scores in two gesture sets; they also had scores within the subset of the best five features in the remaining one. Further, the classification accuracies of four different feature sets presented remarkable increases when FEn and PEn are included as additional features. Outcomes support the use of FEn and PEn for hand gesture description when computational parameters are properly selected, and they could be useful in supporting the development of robotic arms and prostheses myoelectric control

Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking

We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 \u3bcm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle

Mobile elites at Frattesina: flows of people in a Late Bronze Age ‘port of trade’ in northern Italy

Following a mid twelfth-century BC demographic crisis, Frattesina, in northern Italy, arose as a prominent hub linking continental Europe and the Mediterranean, as evidenced by the remarkable variety of exotic materials and commodities discovered at the site. Debate persists, however, about the extent to which migrants influenced the foundation and development of Frattesina. The authors present the results of strontium isotope analyses, which suggest significant migration to the site, particularly of elites, mostly from within a 50km radius. Among these non-indigenous people, the authors identify a \u2018warrior-chief\u2019, whom they interpret as representing a new, more hierarchical society

EMG-Based Characterization of Walking Asymmetry in Children with Mild Hemiplegic Cerebral Palsy

5 May 2019; Accepted: 24 June 2019; Published: 27 June 2019 Abstract: Hemiplegia is a neurological disorder that is often detected in children with cerebral palsy. Although many studies have investigated muscular activity in hemiplegic legs, few EMG-based findings focused on una ected limb. This study aimed to quantify the asymmetric behavior of lower-limb-muscle recruitment during walking in mild-hemiplegic children from surface-EMG and foot-floor contact features. sEMG signals from tibialis anterior (TA) and gastrocnemius lateralis and foot-floor contact data during walking were analyzed in 16 hemiplegic children classified as W1 according to Winter’ scale, and in 100 control children. Statistical gait analysis, a methodology achieving a statistical characterization of gait by averaging surface-EMG-based features, was performed. Results, achieved in hundreds of strides for each child, indicated that in the hemiplegic side with respect to the non-hemiplegic side, W1 children showed a statistically significant: decreased number of strides with normal foot-floor contact; decreased stance-phase length and initial-contact sub-phase; curtailed, less frequent TA activity in terminal swing and a lack of TA activity at heel-strike. The acknowledged impairment of anti-phase eccentric control of dorsiflexors was confirmed in the hemiplegic side, but not in the contralateral side. However, a modified foot-floor contact pattern is evinced also in the contralateral side, probably to make up for balance requirements

A new method for spatial mode shifting of stabilized optical cavities for the generation of dual-color X-rays

We propose an innovative method to shift the transversal position of the focal point of an optical cavity keeping it actively frequency stabilized. Our cavity is a 4 mirrors bow-tie cavity and the spatial shift of the resonant mode is obtained by properly rotating the two curved mirrors by piezo actuators. This method allows us to move the transversal position of the cavity focal point of 135 µm in a time of 50 ms, keeping the resonance condition of the cavity by means of the Pound–Drever–Hall technique. We propose to use this technique for the generation of 2-color X-rays via Inverse Compton Scattering (ICS). This technique exploits the large average power stored in the high finesse cavity by shifting the laser beam with respect to the electron beam trajectory, hence controlling the spatial superposition of the electron and photon beams in the interaction region. Arranging two cavities assembled one on top of the other, with different collision angle with the electron beam, allows the generation of X-ray bursts of different energies just by swiftly moving the two cavities, switching the two focal points onto the electron beam trajectory, thus activating in sequence two different ICS spectral lines

Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin

The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane leaflets. We argue that the present experimental protocol can be applied to the investigation of the supramolecular organization of encapsulated luminescent drugs/molecules all the way from the production phase to their state within living matter

Mast cell disorders, melanoma and pancreatic carcinoma: from a clinical observation to a brief review of the literature.

Mastocytosis can be associated with other clonal or non-clonal hematologic diseases as well as a variety of non-hematologic malignancies. A 75-year-old Caucasian male patient was referred to us with a 5-month history of neutrophilic leukocytosis and mild splenomegaly. He had developed a cutaneous melanoma sixteen years ago. According to the clinical and pathological features, a final diagnosis of systemic mastocytosis was established. The patient started treatment with interferon-α at a dose of 3 MIU/day, combined with low doses of prednisone. We observed a rapid disappearance of symptoms. Unfortunately, after 3 months a diagnosis of pancreatic adenocarcinoma was established. A review of the literature suggests that mastocytes could have a pivotal role in several malignancies. Different chemokines, mitogenic factors, chemical mediators of inflammation, and specific gene mutations could explain the association between mastocytosis and other hematologic and non-hematologic disorders. </p

Observation of CR Anisotropy with ARGO-YBJ

The measurement of the anisotropies of cosmic ray arrival direction provides important informations on the propagation mechanisms and on the identification of their sources. In this paper we report the observation of anisotropy regions at different angular scales. In particular, the observation of a possible anisotropy on scales between $\sim$ 10 $^{\circ}$ and $\sim$ 30 $^{\circ}$ suggests the presence of unknown features of the magnetic fields the charged cosmic rays propagate through, as well as potential contributions of nearby sources to the total flux of cosmic rays. Evidence of new weaker few-degree excesses throughout the sky region $195^{\circ}\leq$ R.A. $\leq 315^{\circ}$ is reported for the first time.Comment: Talk given at 12th TAUP Conference 2011, 5-9 September 2011, Munich, German