Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv)

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75?h, typically between 140 and 160?g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5?% of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0?% boundary for longer periods of time when compared to a traditional proportional–integral–derivative algorithm, which tends to destabilize with increasing cell density.     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.     

Harpellales in the digestive tracts of Ephemeroptera and Plecoptera nymphs from Veracruz, Mexico

This is the first report of Harpellales (Zygomycota) from Mexico, including herein only the endosymbiotic species of gut fungi in the digestive tracts or shed exuviae of Plecopteran and Ephemeropteran nymphs. Four new species are described: Allantomyces zopilotei, Bojamyces olmecensis, Gauthier-omyces viviparus and Graminella ophiuroidea. Among previously known Harpellales, Lancisporomyces nemouridarum and Zygopolaris ephemeridarum are southern range extremes and new records for Mexico. All species are illustrated and discussed relating to biogeographic implications of the new reports from Mexico, as well as the particular environmental circumstances of the Harpellales in the tropics.     

CO2 and O2 dynamics in human-impacted watersheds in the state of São Paulo, Brazil

We studied the spatial and temporal variation in O₂ and dissolved inorganic carbon (DIC) forms concentrations in ten subtropical watersheds located in the state of São Paulo, Brazil, with different degrees of impact by urbanization and land-use changes. Additionally, we used stable carbon isotopic composition of DIC to explain observed patterns. We found that land-cover changes and watershed geology are the main drivers of DIC distribution. Land-cover/use changes influence the riverine DIC in two ways: by replacing the original Cerrado 3 (C3)-type forest vegetation by C4-type vegetation composed of grasses (pasture), and by sugarcane. Most domestic sewage is dumped untreated into rivers in the state of São Paulo. Consequently, in the most densely populated watersheds, sewage is an important source of labile carbon and consequently of DIC to rivers. In terms of geology, although silicate weathering that produces kaolinite is the main type of weathering in the watersheds, the weathering of carbonate cements present in the geological formations of the western portion of the state of São Paulo are also an important source of DIC to rivers.     

Helophyte germination in a Mediterranean salt marsh: Gut‐passage by ducks changes seed response to salinity

Question: In seeds which are regularly consumed by waterbirds in the field, how does gut‐passage modify their response to salinity gradients? Location: Doñana National Park salt marsh, south‐west of Spain. Methods: Seeds of Scirpus litoralis and Scirpus maritimus were collected and force fed to mallards (Anas platyrhynchos). Both the ingested seeds (passage) and non‐ingested seeds (controls) were exposed, in germination chambers, to a salinity range similar to that observed in the field (0–32 dS/m). After 30 days, the total percentage germination, the duration of the dormancy period and the germination speed were computed. The response of the different germination parameters to ingestion and salinity was analyzed using generalized lineal models. Recovery tests on seeds that did not germinate in the various treatments and tests of the effect of ingestion on the intrinsic variability in seed response were also performed. Results: An increase in salinity reduced germinability and increased the length of dormancy, while gut pas sage increased the intrinsic variability of the temporal seed response in both species. In S. litoralis there was a significant interaction between the effects of salinity and passage on germination rate. Passage increased germination rate at low salinities (≤2 dS/m) but decreased it at high salinities (≥4 dS/m). Conclusion: Gut‐passage by ducks significantly changes seed response to salinity. The outcome of plant‐animal interactions can be influenced by environmental gradients. Studies of germination in response to gut passage that do not take such gradients into account may produce misleading results.     

Characterization of NADPH-diaphorase-positive glial cells of the tench optic nerve after axotomy

NADPH-diaphorase (ND) positive cell types were characterized throughout the optic nerve of the tench in normal conditions and after optic nerve transection with survival periods of 1, 3, 7, 14, 30, 60, 120 and 180 days. Astrocytic markers (S100 and glutamine synthetase) and the microglial marker tomato lectin were employed. In the control prechiasmatic optic nerve two types (types I and II) of ND-positive glial cells appeared. All type I cells showed S100 immunoreactivity, whereas only a subpopulation of them were positive to glutamine synthetase. Type II cells only presented S100 immunoreactivity. In the control anterior optic tract, all ND-positive glial cells (type III) presented immunolabeling to S100 and glutamine synthetase. After transection, types I and II did not show any changes in the staining patterns for the glial markers when observed. Two new types of ND-positive glial cells (types IV and V) were observed after axotomy. All type IV cells were S100-immunopositive, and a subpopulation presented glutamine synthetase immunolabeling. Only a subpopulation of type V cells showed glutamine synthetase immunostaining. The presence of type IV or V cells in the lesioned optic nerve occurred simultaneously with significant decreases or absence of type I cells. These data suggest that type I and III cells are astrocytes and type II cells are oligodendrocytes. Types IV and V cells are the result of the activation of type I cells after optic nerve section. The polymorphism observed in ND-positive cells may reflect different cell functions during degenerative and regenerative processes.