Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Involvement of an alternative oxidase in oxidative stress and mycelium-to-yeast differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

Trimethoxy-chalcone derivatives inhibit growth of Leishmania braziliensis: synthesis, biological evaluation, molecular modeling and structure-activity relationship (SAR)

In this work we described the synthesis, the antileishmanial activity and the molecular modeling and structure-activity relationship (SAR) evaluations of a series of chalcone derivatives. Among these compounds, the methoxychalcones 2h, 2i, 2j, 2k and 2l showed significant antileishmanial activity (IC(50)<10 μM). Interestingly 2i (IC(50)=2.7 μM), 2j (IC(50)=3.9 μM) and 2k (IC(50)=4.6 μM) derivatives presented better antileishmanial activity than the control drug pentamidine (IC(50)=6.0 μM). Our SAR study showed the importance of methoxy di-ortho substitution at phenyl ring A and the relationship between the frontier orbital HOMO coefficients distribution of these molecules and their activity. The most active compounds 2h, 2i, 2j, 2k, and 2l fulfilled the Lipinski rule-of-five which theoretically is important for good drug absorption and permeation through biological membranes. The potential profile of 2j (IC(50)=3.9 μM and CC(50)=216 μM) pointed this chalcone derivative as a hit compound to be further explored in antileishmanial drug design.     

Silencing of mitochondrial alternative oxidase gene of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.     

Heterologous expression of mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> rescues impaired growth in <Emphasis Type="Italic">Δndt1Δndt2 Saccharomyces cerevisiae</Emphasis> strain

Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD⁺), generating a large mitochondrial membrane potential. The NAD⁺ carrier utilized the electrochemical proton gradient to drive NAD⁺ entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.     

Comparison of the accumulation of 137Cs and 90Sr by six spring wheat varieties

The uptake of ¹³⁷Cs and ⁹⁰Sr by six varieties of spring wheat (Triticum aestivum) was compared in field trials on land contaminated by the Chernobyl accident. All the experimental varieties are officially adopted for agricultural use in Belarus and are used in large-scale production. Under identical conditions of nutrition, the productivity of the varieties varied significantly by a factor of 1.3. The extent of ¹³⁷Cs and ⁹⁰Sr accumulation by wheat grain, quantified as the concentration ratio, differed between the varieties by as much as a factor of 1.6, for both radionuclides. There was a significant linear positive correlation between the ⁹⁰Sr activity concentration in grain and straw, and the calcium concentration. The correlation between ¹³⁷Cs and potassium was not significant. The results suggest that certain varieties of spring wheat used in normal agricultural practice accumulate less ¹³⁷Cs and ⁹⁰Sr into grain than others. Some spring wheat varieties accumulated relatively less ¹³⁷Cs, but did not accumulate less ⁹⁰Sr. One variety, Quattro, had a significantly lower uptake of both ⁹⁰Sr (for grain) and ¹³⁷Cs (for both grain and straw) than that of the other varieties tested. The reduction efficiency achieved by the use of these varieties, however, is not as high as that achieved by soil amelioration techniques in the past. Nevertheless, since there are no additional costs or production losses associated with these varieties, their use in the contaminated areas is worth considering as a simple, practical, and effective contribution to reducing the uptake of both ⁹⁰Sr and ¹³⁷Cs and allowing farmers to produce food-grade grain.     

Protease domain of human ADAM33 produced by Drosophila S2 cells

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies ( approximately 10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].     

Methods for assessing DNA hybridization of peptide nucleic acid-titanium dioxide nanoconjugates

We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO₂) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA-TiO₂ nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA-TiO₂ nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO₂ nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO₂-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.