292 research outputs found
MOLECULAR DYNAMICS SIMULATIONS OF BIOLOGICAL MACROMOLECULES: APPLICATIONS TO STRUCTURAL VACCINOLOGY AND PEPTIDE DESIGN
This thesis work is splitted into two parts. The first one is about a computational method for epitope predictions on antigenic proteins, while the second one is related to the characterization of folding/unfolding processes of small natural polypeptides. Starting with the first topic, an increasing number of functional studies of proteins have shown that sequence and structural similarities alone
may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific
antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties
of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we
investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify
nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on
the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the
protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations
with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes,
it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative
binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures
based only on physical properties. A public web server (BEPPE) has been implemented with MLCE method in order to make it available to the scientific community. Changing topic to folding/unfolding, the analysis of the folding
mechanism in peptides adopting well defined
secondary structure is fundamental
to understand protein folding.
Herein, we describe the thermal unfolding
of two 15-mer polypeptides (called QK and QK-L10A) homologue to the vascular endothelial
growth factor binding region. In particular, on the basis of the temperature dependencies, we characterize the molecules through the combination
of spectroscopic (CD and NMR) and computational
analyses (MD) highlighting their folding/unfolding steps and how these structures can be used in peptide design
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Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.
Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions
In vitro effects of simulated microgravity on Sertoli cell function
With the advent of space flights questions concerning the effects of microgravity (0G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1G samples. Initially, 0G seemed to disturb antioxidant pro- tection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate pro- duction at 0G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.With the advent of space flights questions concerning the effects of microgravity (0G) on human reproductive physiology have
13 received great attention. The aim of this study was to evaluate the influence of 0G on Sertoli cells. A Sertoli cell line from mouse testis
14 (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant
15 content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a
16 three-dimensional Random Positioning Machine (3D-RPM); static controls (1G) were positioned on the supporting frame. At the
17 end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium
18 lactate measurements as needed. At 0G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable
19 already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the
20 first 24 h but significantly decreased at 48 h. No changes occurred in the 1G samples. Initially, 0G seemed to disturb antioxidant pro-
21 tection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate pro-
22 duction at 0G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ
23 cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy
24 increase
Demonstration of Universal Parametric Entangling Gates on a Multi-Qubit Lattice
We show that parametric coupling techniques can be used to generate selective
entangling interactions for multi-qubit processors. By inducing coherent
population exchange between adjacent qubits under frequency modulation, we
implement a universal gateset for a linear array of four superconducting
qubits. An average process fidelity of is estimated for
three two-qubit gates via quantum process tomography. We establish the
suitability of these techniques for computation by preparing a four-qubit
maximally entangled state and comparing the estimated state fidelity against
the expected performance of the individual entangling gates. In addition, we
prepare an eight-qubit register in all possible bitstring permutations and
monitor the fidelity of a two-qubit gate across one pair of these qubits.
Across all such permutations, an average fidelity of
is observed. These results thus offer a path to a scalable architecture with
high selectivity and low crosstalk
Epidemiological characteristics and diagnostic approach in patients admitted to the emergency room for transient loos of consciousness: Group for Syncope Study in the Emergency Room (GESINUR) study
Aims: To assess the clinical presentation and acute management of patients with transient loss of consciousness (T-LOC) in the emergency department (ED).
Methods and results: A multi-centre prospective observational study was carried out in 19 Spanish hospitals over 1 month. The patients included were 14 years old and were admitted to the ED because of an episode of T-LOC. Questionnaires and corresponding electrocardiograms (ECGs) were reviewed by a Steering Committee (SC) to unify diagnostic criteria, evaluate adherence to guidelines, and diagnose correctly the ECGs. We included 1419 patients (prevalence, 1.14%).ECG was performed in 1335 patients (94%) in the ED: 498 (37.3%) ECGs were classified as abnormal. The positive diagnostic yield ranged from 0% for the chest X-ray to 12% for the orthostatic test. In the ED, 1217 (86%) patients received a final diagnosis of syncope, whereas the remaining 202 (14%) were diagnosed of non-syncopal transient lossof consciousness (NST-LOC). After final review by the SC, 1080 patients (76%) were diagnosed of syncope, whereas 339 (24%) were diagnosed of NST-LOC (P , 0.001). Syncope was diagnosed correctly in 84% of patients. Only 25% of patients with T-LOC were admitted to hospitals.
Conclusion Adherence to clinical guidelines for syncope management was low; many diagnostic tests were performed with low diagnostic yield. Important differences were observed between syncope diagnoses at the ED and by SC decision
Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation
Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate
neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in
flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects.By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1
maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring
cell death, and it enables targeting by motor neurons. We show that Spa¨ tzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases
FOLFOX6 and bevacizumab in non-optimally resectable liver metastases from colorectal cancer
BACKGROUND:
In patients with colorectal liver metastases (CLM) R0 resection significantly improves overall survival (OS).
METHODS:
In this report, we present the results of a phase II trial of FOLFOX6+bevacizumab in patients with non-optimally resectable CLM. Patients received six cycles of FOLFOX6+ five of bevacizumab. Patients not achieving resectability received six additional cycles of each. A PET-CT was performed at baseline and again within 1 month after initiating treatment.
RESULTS:
From September 2005 to July 2009, 21 patients were enrolled (Male/Female: 15/6; median age: 65 years). An objective response (OR) was documented in 12 cases (57.1%; complete responses (CRs): 3, partial response (PR): 9); one patient died from toxicity before surgery. Thirteen patients underwent radical surgery (61.9%). Three (23%) had a pathological CR (pCR). Six patients (46.1%) experienced minor postsurgical complications. After a median 38.8-month follow-up, the median OS was 22.5 months. Patients achieving at least 1 unit reduction in Standard uptake value (SUV)max on PET-CT had longer progression-free survival (PFS) (median PFS: 22 vs 14 months, P=0.001).
CONCLUSIONS:
FOLFOX6+bevacizumab does not increase postsurgical complications, yields high rates of resectability and pCR. Early changes in PET-CT seem to be predictive of longer PFS
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