A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption

A two-stage ultrafiltration process was applied to the aqueous phase of Tetraselmis suecica after breaking its cell wall by high-pressure homogenization. Microscopic observation revealed that the cells were completely disrupted from 600 bar and cell fragmentation of the cells was also noticeable after 800 bar. In addition, the highest concentration of all the molecules of interest in the aqueous phase was observed at 1,000 bar and a temperature of 46 °C while preserving the integrity of the molecules of interest in the downstream process. After centrifugation, the aqueous phase was submitted to ultrafiltration through two consecutive membranes of different molecular weight cutoffs. Complete retention of starch was possible with a 100-kDa membrane and separation of sugars from proteins with a 10-kDa membrane on the remaining mixture. After testing the process with model solutions, the transmembrane pressure selected was 2.07 bar, which succeeded in retaining starch and pigments during the first part of the process, and proteins during the second part. A linear correlation between the permeate flux rate and the pressure was observed in both parts of the process.     

NMR structural studies of interactions of a small, nonpeptidyl Tpo mimic with the thrombopoietin receptor extracellular juxtamembrane and transmembrane domains

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.     

Differential impact of glucose levels and advanced glycation end-products on tubular cell viability and pro-inflammatory/profibrotic functions

High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally used to mimic diabetes in cellular models. Both models have shown an increase of apoptosis, oxidative stress and pro-inflammatory cytokine production in tubular cells. However, the impact of the two conditions combined has rarely been studied. In addition, the impact of glucose level variation due to cellular consumption is not clearly characterized in such experiments. Therefore, the aim of this study was to compare the effect of HG and AGE separately and of both on tubular cell phenotype changes in the HK2 cell line. Moreover, glucose consumption was monitored every hour to maintain the glucose level by supplementation throughout the experiments. We thus observed a significant decrease of apoptosis and H₂O₂ production in the HK2 cell. HG or AGE treatment induced an increase of total and mitochondrial apoptosis as well as TGF-β release compared to control conditions; however, AGE or HG led to apoptosis preferentially involving the mitochondria pathway. No cumulative effect of HG and AGE treatment was observed on apoptosis. However, a pretreatment with RAGE antibodies partially abolished the apoptotic effect of HG and completely abolished the apoptotic effect of AGE. In conclusion, tubular cells are sensitive to the lack of glucose as well as to the HG and AGE treatments, the AGE effect being more deleterious than the HG effect. Absence of a potential synergistic effect of HG and AGE could indicate that they act through a common pathway, possibly via the activation of the RAGE receptors.     

Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.     

Field evidence for a proximate role of food shortage in the regulation of hibernation and daily torpor: a review

Hibernation and daily torpor (heterothermy) have long been assumed to be adaptive responses to seasonal energy shortage. Laboratory studies have demonstrated that food shortage alone can trigger the use of heterothermy. However, their potential to predict heterothermic responses in the wild is limited, and few field studies demonstrate the dependence of heterothermy on food availability under natural conditions. Thus, the view of heterothermy as an energy saving strategy to compensate for food shortage largely remains an untested hypothesis. In this paper, we review published evidence on the proximate role of food availability in heterothermy regulation by endotherms, and emphasize alternative hypotheses that remain to be tested. Most studies have relied on correlative evidence. Manipulations of food availability, that demonstrate the proximate role of food availability, have been conducted in only five free-ranging heterotherms. Several other metabolic constraints covary with food availability and can confound its effect. Shortage in water availability, the nutritional composition of food, or subsequent conversion of food in fat storage all could be actual proximate drivers of heterothermy regulation, rather than food shortage. Social interactions, competition for food and predation also likely modulate the relative strength of food shortage between individuals. The ecological relevance of the dependence of heterothermy on food availability remains to be assessed in field experiments that account for the confounding effects of covarying environmental and internal factors.     

Reproductive resilience to food shortage in a small heterothermic primate

The massive energetic costs entailed by reproduction in most mammalian females may increase the vulnerability of reproductive success to food shortage. Unexpected events of unfavorable climatic conditions are expected to rise in frequency and intensity as climate changes. The extent to which physiological flexibility allows organisms to maintain reproductive output constant despite energetic bottlenecks has been poorly investigated. In mammals, reproductive resilience is predicted to be maximal during early stages of reproduction, due to the moderate energetic costs of ovulation and gestation relative to lactation. We experimentally tested the consequences of chronic-moderate and short-acute food shortages on the reproductive output of a small seasonally breeding primate, the grey mouse lemur (Microcebus murinus) under thermo-neutral conditions. These two food treatments were respectively designed to simulate the energetic constraints imposed by a lean year (40% caloric restriction over eight months) or by a sudden, severe climatic event occurring shortly before reproduction (80% caloric restriction over a month). Grey mouse lemurs evolved under the harsh, unpredictable climate of the dry forest of Madagascar and should thus display great potential for physiological adjustments to energetic bottlenecks. We assessed the resilience of the early stages of reproduction (mating success, fertility, and gestation) to these contrasted food treatments, and on the later stages (lactation and offspring growth) in response to the chronic food shortage only. Food deprived mouse lemurs managed to maintain constant most reproductive parameters, including oestrus timing, estrogenization level at oestrus, mating success, litter size, and litter mass as well as their overall number of surviving offspring at weaning. However, offspring growth was delayed in food restricted mothers. These results suggest that heterothermic, fattening-prone mammals display important reproductive resilience to energetic bottlenecks. More generally, species living in variable and unpredictable habitats may have evolved a flexible reproductive physiology that helps buffer environmental fluctuations.