A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions Preserves the Chemical Characteristics and Biological Activity of Lyo-Secretome Isolated by Ultrafiltration

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.     

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

Myoclonic absence-like seizures and chromosome abnormality syndromes

PURPOSE: We explored the relationship between myoclonic absence seizures (MAS) and underlying chromosome disorders. METHODS: Among 14 patients with MAS observed in three centers, 5 had typical cryptogenic myoclonic absence epilepsy (MAE), 2 had MAS associated with other seizure types (1 with signs of a neuronal migration abnormality and 1 with signs of a metabolic disorder), and 7 had MAS, with or without other seizure types, complicating a chromosome abnormality syndrome-2 with trisomy 12p, 4 with Angelman syndrome, and 1 with inv dup (15). RESULTS: In the 7 patients with chromosomopathy, MAS appeared at a mean age of 2.9 years (range 4 months to 6 years 6 months), had a duration of 4-20 s, and were accompanied by reduced awareness and rhythmic myoclonic jerks involving proximal limb muscles. Ictal EEG showed 2- to 3-Hz generalized spike-and-wave discharges. CONCLUSIONS: In these patients, MAS differed slightly from those of typical MAE: age of onset was earlier, absences were of shorter duration, and no clear increase in muscular tone was noted. Abnormal expression of genes codifying for the subfamily of K+ channels and for gamma-aminobutyric acid-3 subunit receptors (GABRB3), both located in the chromosome segments involved in the chromosomopathies presented by our patients, could be responsible for the same generalized seizure type. Chromosome analysis should be performed in patients with mental retardation and MAS, especially when the ictal pattern does not completely overlap that observed in MAE.     

Focus of B-to-B e-commerce initiatives and related benefits in manufacturing small- and medium-sized enterprises

Empirical research into business-to-business e-commerce issues involving manufacturing small- and medium-sized enterprises (SMEs) is still embryonic. In an attempt to partially fill this gap, this paper presents empirical data from an electronic survey conducted among 96 manufacturing SMEs to investigate e-commerce initiatives and their related benefits. E-commerce initiatives are assessed using a set of 36 business processes that can be conducted electronically. These processes were classified according to their focus: customer (downstream), supplier (upstream) or in-house. The research findings point to four main profiles of manufacturing SMEs with different e-commerce focuses. The first group seems to lack any focus or may still be exploring e-commerce opportunities. The second and third groups are supplier- and customer-focused, respectively. The fourth group consists of the more involved SMEs that have leveraged their e-commerce initiatives with both their customers and their suppliers. Results also suggest the existence of a close alignment between e-commerce focus and related benefits.An earlier and much shorter version of this paper was published in the Proceedings of the 37th Hawaii International Conference on System Sciences (HICSS-37). Helpful comments from two anonymous reviewers were greatly appreciated. The authors gratefully acknowledge financial support from SSHRC, FCAR and NSERC.     

Assembling a lasing hybrid material with supramolecular polymers and nanocrystals

The combination of bottom-up and top-down processes to organize nanophases in hybrid materials is a key strategy to create functional materials. We found that oxide and sulphide nanocrystals become spontaneously dispersed in organic media during the self-assembly of nanoribbon supramolecular polymers. These nanoribbon polymers form by self-assembly of dendron rodcoil molecules, which contain three molecular blocks with dendritic, rod-like, and coil-like architectures. In an electric field these supramolecular assemblies carrying bound nanocrystals migrate to the positive electrode in an etched channel and align in the field. In the system containing ZnO nanocrystals as the inorganic component, both phases are oriented in the hybrid material forming an ultraviolet lasing medium with a lower threshold relative to pure ZnO nanocrystals.     

Performance Analysis of a DCPSK Up-Link for a Regenerative Satellite Repeater

A mixed analytic and simulation method is described for the study of a DCPSK up-link used in a regenerative satellite transmission system. A great variety of impairments is considered and their effects on error probability are evaluated, thus emphasizing the performance evaluation in a "real situation." A case study is worked out in detail to stress the design-oriented approach.     

Novel polyurethane‐based thermosensitive hydrogels as drug release and tissue engineering platforms: design and in vitro characterization

Poloxamer P407 (P407) is a Food and Drug Administration approved triblock copolymer; its hydrogels show fast dissolution in aqueous environment and weak mechanical strength, limiting their in vivo application. In this work, an amphiphilic poly(ether urethane) (NHP407) was synthesized from P407, an aliphatic diisocyanate (1,6‐hexanediisocyanate) and an amino acid derived diol (N‐Boc serinol). NHP407 solutions in water‐based media were able to form biocompatible injectable thermosensitive hydrogels with a lower critical gelation temperature behavior, having lower critical gelation concentration (6% w/v versus 18% w/v), superior gel strength (G′ at 37 °C about 40 000 Pa versus 10 000 Pa), faster gelation kinetics (<5 min versus 15–30 min) and higher stability in physiological conditions (28 days versus 5 days) compared to P407 hydrogels. Gel strength and PBS absorption at 37 °C increased whereas dissolution rate (in phosphate‐buffered saline (PBS) at 37 °C) and permeability to nutrients (studied using fluorescein isothiocyanate–dextran model molecule) decreased as a function of NHP407 hydrogel concentration from 10% to 20% w/v. By varying the concentration, NHP407 hydrogels were thus prepared with different properties which could suit specific applications, such as in situ drug/cell delivery or bioprinting of scaffolds. Moreover, deprotected amino groups in NHP407 could be exploited for the grafting of bioactive molecules obtaining biomimetic hydrogels. © 2016 Society of Chemical Industry     

Effect of the chemical nature and concentration of palladium (II) precursors on the palladium deposition by atomic hydrogen electrochemically assisted

Pd deposits are obtained by applying −1.92 mA cm⁻² during 180 min, employing 0.06 M PdCl₂ + 1 M HCl and 0.06 M PdCl₂ + 28% NH₃ solutions. Some features on Pd depositions using x M PdCl₂ + 1 M HCl (x = 0.130, 0.060, 0.030, 0.025, 0.020 and 0.010) solutions are detailed. Based on roughness factor (S) and deposition efficiency (?_Pd) values, Pd deposition is more efficient using PdCl₂ + 1 M HCl than PdCl₂ + 28% NH₃ (?_Pd = 99%, S = 6 in 1 M HCl vs. ?_Pd = 17%, S = 3 in 28% NH₃). A highly rough Pd deposit (S = 357) is obtained, by applying −1.92 mA cm⁻² during 180 min, using a 0.020 M PdCl₂ + 1 M HCl solution.