Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro‐patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi‐nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer‐by‐layer (LbL) polyelectrolyte multilayer deposition was combined with a micro‐molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self‐assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4‐styrene‐sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly‐L ‐arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅‐coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586–596. © 2012 Wiley Periodicals, Inc.     

Analysis of PIN1 WW domain through a simple statistical mechanics model

We have applied a simple statistical mechanics Go-like model to the analysis of the PIN1 WW domain, resorting to mean field and Monte Carlo techniques to characterize its thermodynamics, and comparing the results with the wealth of available experimental data. PIN1 WW domain is a 39-residue protein fragment which folds on an antiparallel beta-sheet, thus representing an interesting model system to study the behavior of these secondary structure elements. Results show that the model correctly reproduces the two-state behavior of the protein, and also the trends of the experimental phi(T) values. Moreover, there is a good agreement between Monte Carlo results and the mean field ones, which can be obtained with a substantially smaller computational effort.     

Compartment-Specific Biosensors Reveal a Complementary Subcellular Distribution of Bioactive Furin and PC7

1. Download high-res image (218KB)
2. Download full-size image

     

Multiple freshwater invasions of the tapertail anchovy (Clupeiformes: Engraulidae) of the Yangtze River

Freshwater fish evolved from anadromous ancestors can be found in almost all continents. The roles of paleogeographic events and nature selection in speciation process often are under focus of research. We studied genetic diversity of anadromous and resident tapertail anchovies (Coilia nasus species complex) in the Yangtze River Basin using 4,434 nuclear loci, and tested the history of freshwater invasion of C. nasus. We found that both C. brachygnathus and C. nasus were valid species, but the resident C. nasus taihuensis and the anadromous C. nasus were not different genetically based on Bayes factor species delimitation (BFD*). Maximum likelihood tree, Network, PCA and STRUCTURE analyses all corroborated the results of BFD*. Two independent freshwater invasion events of C. nasus were supported, with the first event occurring around 4.07 Ma and the second happened around 3.2 Ka. The time of the two freshwater invasions is consistent with different paleogeographic events. Estimation showed that gene flow was higher within ecotypes than between different ecotypes. F‐DIST analyses identified 120 disruptive outliers by comparing C. brachygnathus to anadromous C. nasus, and 21 disruptive outliers by comparing resident C. nasus to anadromous C. nasus. Nine outliers were found to be common between the two comparisons, indicating that independent freshwater invasion of C. nasus might involve similar molecular pathways. The results of this study suggest that adaptation to landlocked freshwater environment of migratory fish can evolve multiple times independently, and morphology of landlocked ecotypes may cause confusion in their taxonomy.     

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.     

Replication Banding in two Mediterranean Moray eels: Chromosomal Characterization and Comparison

Early and late replication bandings have been obtained by in vitro BrdU incorporation in the Mediterranean Muraenidae species Muraena helena and Gymnothorax unicolor, and used to characterize their karyotypes. A comparative analysis of the banding patterns allowed to point out high karyotype similarity as well as chromosome rearrangements that occurred in karyotype evolution between these species.     

Evolution of ragweed pollen concentrations, sensitization and related allergic clinical symptoms in Parma (northern Italy)

The pollen released in the atmosphere by the ragweed represents a question of public health in several European countries. In Italy, the ragweed is mostly distributed in the North. In our region (Emilia Romagna), the presence of ragweed was not described yet if not occasionally, but this plant is thriving well in the North of the Po river. The aim of our study was to estimate the concentration trends of ragweed pollens in the air of Parma starting from 1992 until 2008 and to describe the clinical related situation. The aerobiological surveillance was made with the methods standardized by the Italian Association of Aerobiology. We analyzed 19,468 outpatients affected by respiratory disease. The patients studied address our clinical Center, mainly with pathologies respiratory, most of them with allergic origin. To detect the existence of significant trends and correlations since, we used the non-parametric tests with SPSS software. Our observations showed that since 1995, the year until when pollens of ragweed were only sporadically observed in the air of Parma, there has been a significant increase in ragweed pollens. Among the patients addressed at our clinical Center, 876 patients had positive SPT (skin prick test) for ragweed pollen with respiratory illnesses, all polysensitized. Besides, we found a significant increase in patients with positive SPT for the ragweed, and among these, the increase in the asthma has been significant. On the basis of our results, we expect, in the absence of intervention from public authorities, a more significant increase in the positive subjects and an aggravation of the symptoms related to the presence of ragweed pollen in the air of Parma.     

Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences

The evolutionary pathway of Palinuridae (Crustacea, Decapoda) is still controversial, uncertain and unexplored, expecially from a karyological point of view. Here we describe the South African spiny lobster Jasus lalandii karyotype: n and 2n values, heterochromatin distribution, nucleolar organizer region (NOR) location and telomeric repeat structure and location. To compare the genomic and chromosomal organization in Palinuridae we located NORs in Panulirus regius, Palinurus gilchristi and Palinurus mauritanicus: all species showed multiple NORs. In J. lalandii NORs were located on three chromosome pairs, with interindividual polymorphism. In P. regius and in the two Palinurus species NORs were located on two chromosome pairs. In the two last species 45S ribosomal gene loci were also found on B chromosomes. In addition, the nature and location of telomeric repeats were investigated by FISH in J. lalandii, P. gilchristi, P. mauritanicus Palinurus elephas, and P. regius (Palinuridae, Achelata), and in Scyllarus arctus (Scyllaridae, Achelata): all these Achelata species showed the (TTAGG)n pentameric repeats. Furthermore, in J. lalandii these repeats occurred in all the telomeres and in some interstitial chromosomal sites, associated with NORs.     

Synthesis, crystal structure, spectral properties and cytotoxic activity of platinum(II) complexes of 2-acetyl pyridine and pyridine-2-carbaldehyde N(4)-ethyl-thiosemicarbazones

The reactions of Na2PtCl4 with pyridine-2-carbaldehyde and 2-acetyl pyridine N(4)-ethyl-thiosemicarbazones, HFo4Et and HAc4Et respectively, afforded the complexes [Pt(Fo4Et)Cl], [Pt(HFo4Et)2]Cl2, [Pt(Fo4Et)2] and [Pt(Ac4Et)Cl], [Pt(HAc4Et)2]Cl2 x 2H2O, [Pt(Ac4Et)2]. The new complexes have been characterized by elemental analyses and spectroscopic studies. The crystal structure of the complex [Pt(Ac4Et)Cl] has been solved. The anion of Ac4E coordinates in a planar conformation to the central platinum(II) through the pyridyl N, azomethine N and thiolato S atoms. Intermolecular hydrogen, non-hydrogen bonds, pi-pi and weak Pt-pi contacts lead to aggregation and a supramolecular assembly. The cytotoxic activity for the platinum(II) complexes in comparison to that of cisplatin and thiosemicarbazones was evaluated in a pair of cisplatin-sensitive and -resistant ovarian cancer cell lines A2780 and A2780/Cp8. The platinum(II) complexes showed a cytotoxic potency in a very low micromolar range and were found able to overcome the cisplatin resistance of A2780/Cp8 cells.     

A monolithic silicon based integrated signal generation and detection system for monitoring DNA hybridisation

The aim of this work was to develop an integrated solution to DNA hybridisation monitoring for diagnostics on a monolithic silicon platform. A fabrication process was developed incorporating a gold initiation electrode patterned directly onto a PIN photodiode detector. Patterned interdigitated type electrodes exhibited the smallest reduction in photodiode sensitivity, therefore these were chosen as the ECL initiator design. A novel DNA hybridisation assay was developed based on the displacement of a partially mismatched complementary strand by a perfectly matched labelled complementary strand. Pre-hybridised thiolated oligonucleotide and unlabelled 25% mismatched oligonucleotide were assembled on the gold initiation electrode. On addition of the labelled perfectly complementary oligonucleotide, the mismatched strands were displaced and a signal was generated. The sensitivity of the photodiode to light emitted at 620 nm, the ruthenium emission wavelength, was determined and subsequently, the diode current response to light generated by flow addition of ruthenium solution was found to be measurable to a concentration of 10 fM. Pre-hybridised duplex DNA, consisting of thiolated oligonucleotide and ruthenium labelled complementary oligonucleotide, was assembled on the gold initiation electrode. The difference between the current measured during flow of buffer and the ECL co-reactant TPA was three orders of magnitude, indicating that DNA assembled on the surface comprised sufficient ruthenium to generate a measurable signal. Finally, the displacement of unlabelled partial mismatch oligonucleotide from the sensor surface was monitored on addition of the ruthenium labelled perfectly complementary oligonucleotide in TPA flow and the measured photodiode current response was up to 50 times greater.