Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.     

Revisiting the influence of learning in predator functional response,how it can lead to shapes different from type III

Predator/parasitoid functional response is one of the main tools used to study predation behavior, and in assessing the potential of biological control candidates. It is generally accepted that predator learning in prey searching and manipulation can produce the appearance of a type III functional response. Holling proposed that in the presence of alternative prey, at some point the predator would shift the preferred prey, leading to the appearance of a sigmoid function that characterized that functional response. This is supported by the analogy between enzyme kinetics and functional response that Holling used as the basis for developing this theory. However, after several decades, sigmoidal functional responses appear in the absence of alternative prey in most of the biological taxa studied. Here, we propose modeling the effect of learning on the functional response by using the explicit incorporation of learning curves in the parameters of the Holling functional response, the attack rate (a), and the manipulation time (h). We then study how the variation in the parameters of the learning curves causes variations in the shape of the functional response curve. We found that the functional response product of learning can be either type I, II, or III, depending on what parameters act on the organism, and how much it can learn throughout the length of the study. Therefore, the presence of other types of curves should not be automatically associated with the absence of learning. These results are important from an ecological point of view because when type III functional response is associated with learning, it is generally accepted that it can operate as a stabilizing factor in population dynamics. Our results, to the contrary, suggest that depending on how it acts, it may even be destabilizing by generating the appearance of functional responses close to type I.     

Crystal structure and magnetic properties of lanthanide-trans-2-butenoate polymers (Ln = La, Pr, Dy, Ho)

Four lanthanide coordination polymers formulated as {[La₃L₉(H₂O)₃]EtOH · H₂O}_n1, {[Pr₂L₆(H₂O)₂]HL · H₂O}_n2, {[Dy₂L₆(H₂O)]0.5HL · H₂O}_n3 and {[Ho₂L₆(H₂O)]0.5HL · H₂O}_n4 and HL = trans-2-butenoic acid have been synthesized from the corresponding pure lanthanide oxide and HL acid in water at pH 3. The compounds were characterized by chemical analysis, IR spectroscopy, thermogravimetry, variable-temperature magnetic susceptibility and single crystal X-ray diffraction studies. A common feature in these materials is the presence of two differentiated lanthanide nodes linked by carboxylates into extended chains. These are further connected by inter- and intra-molecular hydrogen-bonds involving ligand and solvate molecules. Weak ferromagnetic interactions appear to be operative in the Dy material.     

Lipoxins in gastric mucosal health and disease

Lipoxins have well characterized anti-inflammatory properties. In recent years, lipoxin A4 and its epimeric counterpart, which is synthesized via aspirin-acetylated cyclooxygenase-2, have been shown to exert very potent protective effects in the stomach. Indeed, suppression of aspirin-triggered lipoxin synthesis, through co-administration of a selective COX-2 inhibitor, results in a significant exacerbation of gastric injury. The gastroprotective effects of lipoxin A4 appear to be receptor mediated, and may be attributable to the ability of this agent to suppress leukocyte adherence to the vascular endothelium and to elevate gastroduodenal blood flow. These effects may be mediated via lipoxin-induced nitric oxide generation. Lipoxins activate a receptor that can also be activated by annexin-1, another substance involved in resolution of inflammation and gastroprotection.     

Lymphatic transport of dietary cholesterol oxidation products,cholesterol and triacylglycerols in rats

Rats were fed on a diet containing 0.5% cholesterol oxidation products (oxysterols) or 0.5% cholesterol for 30 min, and their lymph was collected for 7 h. The amount of each of the individual oxysterols absorbed in the lymph depended on the ingested amounts, but the recovery was the highest for 5alpha,6alpha-epoxycholesterol (10.5%), this being followed by 7-ketocholesterol (5.8%), cholestanetriol (5.2%), 7beta-hydroxycholesterol (4.8%), 7alpha-hydroxycholesterol (3.4%), 5beta,6beta-epoxycholesterol (2.2%), and 25-hydroxycholesterol (1.8%). A diet enriched with oxysterol, but not cholesterol, resulted in increased transport of triacylglycerols in the lymph. These results suggest that the absorption rate of oxysterols depends on the type, and indicate that the effect of dietary oxysterols on the lymphatic transport of triacylglycerols differs from that of dietary cholesterol. It therefore remains to be determined which oxysterol was responsible for the triacyglycerol transport.     

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C₀t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.     

Production of conidia by entomopathogenic fungi: from inoculants to final quality tests

Demand for biopesticides is growing due to the increase of areas under integrated pest management worldwide. Conidia from entomopathogenic fungi play a major role as infective units in the current market of biopesticides. Success in a massive production of fungal conidia include the use of proper long-term conservation microbial methods, aimed at preserving the phenotypic traits of the strains. The development of suitable inoculants should also be considered since that favours a rapid germination and invasiveness of the substrate in solid state cultures (SSC). After the selection of a suitable fungal strain, proven optimization approaches for SSC mainly include the combination of substrates, moisture, texturizers, aeration and moderate stress to induce conidiation. Nonetheless, during storage and upon application in open fields, conidia either as free propagules or imbibed in formulations are subjected to stress due to abiotic factors, then quality should be preserved to resist such harsh conditions. All of these topics are analysed in this report.     

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

Radial growth response to climate change along the latitudinal range of the world's southernmost conifer in southern South America

Aim

We examined whether and how tree radial‐growth responses to climate have changed for the world's southernmost conifer species throughout its latitudinal distribution following rapid climate change in the second half of the 20th century.

Location

Temperate forests in southern South America.

Methods

New and existing tree‐ring radial growth chronologies representing the entire latitudinal range of Pilgerodendron uviferum were grouped according to latitude and then examined for differences in growth trends and non‐stationarity in growth responses to a drought severity index (scPDSI) over the 1900–1993 AD period and also before and after significant shifts in climate in the 1950s and 1970s.

Results

The radial‐growth response of P. uviferum climate was highly variable across its full latitudinal distribution. There was a long‐term and positive association between radial growth and higher moisture at the northern and southern edges of the distribution of this species and the opposite relationship for the core of its distribution, especially following the climatic shifts of the 1950s and 1970s. In addition, non‐stationarity in moisture‐radial growth relationships was observed in all three latitudinal groups (southern and northern edges and core) for all seasons during the 20th century.

Main conclusions

Climate shifts in southern South America in the 1950s and 1970s resulted in different responses in the mean radial growth of P. uviferum at the southern and northern edges and at the core of its range. Dendroclimatic analyses document that during the first half of the 20th century climate‐growth relationships were relatively similar between the southern and northern range edges but diverged after the 1950s. Our findings imply that simulated projections of climate impacts on tree growth, and by implication on forest ecosystem productivity, derived from models of past climate‐growth relationships need to carefully consider different and non‐stationarity responses along the wide latitudinal distribution of this species.