Cell culture monitoring via an auto‐sampler and an integrated multi‐functional off‐line analyzer

Mammalian cell‐based bioprocesses are used extensively for production of therapeutic proteins. Off‐line monitoring of such cultivations via manual sampling is often labor‐intensive and can introduce operator‐dependent error into the process. An integrated multi‐functional off‐line analyzer, the BioProfile FLEX (NOVA Biomedical, Waltham MA) has been developed, which combines the functionality of three off‐line analyzers (a cell counter, an osmometer, and a gas/electrolyte & nutrient/metabolite bio‐profile analyzer) into one device. In addition, a novel automated sampling system has also been developed that allows the BioProfile FLEX to automatically analyze the culture conditions in as many as ten bioreactors. This is the first report on the development and function of this integrated analyzer and an auto‐sampler prototype for monitoring of mammalian cell cultures. Evaluation of the BioProfile FLEX was conducted in two separate laboratories and involved two BioProfile FLEX analyzers and two sets of reference analyzers (Nova BioProfile 400, Beckman‐Coulter Vi‐Cell AS, and Advanced Instruments Osmometer 3900), 13 CHO cell lines and over 20 operators. In general, BioProfile FLEX measurements were equivalent to those obtained using reference analyzers, and the auto‐sampler did not alter the samples it provided to the BioProfile FLEX. These results suggest that the system has the potential to dramatically reduce the manual labor involved in monitoring mammalian cell bioprocesses without altering the quality of the data obtained, and integration with a bioreactor control system will allow feedback control of parameters previously available only for off‐line monitoring. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Apparent vector‐mediated parent‐to‐offspring transmission in an avian malaria‐like parasite

Parasite transmission strategies strongly impact host–parasite co‐evolution and virulence. However, studies of vector‐borne parasites such as avian malaria have neglected the potential effects of host relatedness on the exchange of parasites. To test whether extended parental care in the presence of vectors increases the probability of transmission from parents to offspring, we used high‐throughput sequencing to develop microsatellites for malaria‐like Leucocytozoon parasites of a wild raptor population. We show that host siblings carry genetically more similar parasites than unrelated chicks both within and across years. Moreover, chicks of mothers of the same plumage morph carried more similar parasites than nestlings whose mothers were of different morphs, consistent with matrilineal transmission of morph‐specific parasite strains. Ours is the first evidence of an association between host relatedness and parasite genetic similarity, consistent with vector‐mediated parent‐to‐offspring transmission. The conditions for such ‘quasi‐vertical’ transmission may be common and could suppress the evolution of pathogen virulence.     

Large‐Sized Few‐Layer Graphene Enables an Ultrafast and Long‐Life Aluminum‐Ion Battery

To develop high‐power and high‐energy batteries with a long life remains a great challenge, even combining the benefits of metal (fast kinetics and high capacity) and carbon materials (robust structure). Among them, Al‐ion batteries based on aluminum anode and graphite carbon cathode have gained lots of interests as one of the most promising technologies. Here, it is demonstrated that the size of graphitic material in ab plane and c direction plays an important role in anion intercalation chemistry. Sharply decreasing the size of vertical dimension (c direction) strongly facilitates the kinetics and charge transfer of anions (de)intercalation. On the other hand, increasing the size of horizontal dimension (ab plane) contributes to improving the flexibility of graphitic materials, which results in raising the cycling stability. Meanwhile, chloroaluminate anions are reversibly intercalated into the interlayer of graphite materials, leading to the staging behaviors. In the end, an ultrafast Al‐ion battery with exceptional long life is achieved based on large‐sized few‐layer graphene as a cathode and aluminum metal as an anode.     

Oxidative balance in birds: an atoms‐to‐organisms‐to‐ecology primer for ornithologists

All air‐breathing organisms must face the challenge of oxidative damage, and understanding how animals cope can lend insight into their ecology. Unlike other vertebrates, birds rely primarily on fats to fuel endurance exercise such as migration, and therefore face a greater potential for damage from the reactive by‐products of their own metabolism. We review the physiological ecology of migrating birds through the lens of oxidation–reduction chemistry, underscoring how oxidative balance in wild birds may affect their dietary choices and use of critical stopover habitats during migration. Recent studies reveal that migratory birds prepare for oxidative challenges either by up‐regulating endogenous antioxidants or by consuming them in their diet, and they repair oxidative damage after long flights, although much remains to be discovered about how birds maintain oxidative balance over the course of migration. We conclude by describing some of the most used and useful measures of antioxidant status and oxidative damage that field ornithologists can include in their tool kit of techniques to probe the oxidative balance of wild birds.     

Optimized small‐molecule pull‐downs define MLBP1 as an acyl‐lipid‐binding protein

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC‐MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co‐purifies with wild‐type PYL5 but not a binding site mutant. The K_d of PYL5 for ABA is 1.1 μm , which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain‐related proteins, which resulted in the identification of ligands that co‐purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co‐purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.     

Quallen an Nord‐ und Ostseeküste

Jellyfishes in the North and the Baltic Sea Jellyfish masses are not only a nuisance for bathers but have significant negative effects on marine ecosystems and economy. The causes of worldwide increasing jellyfish mass occurrences are not fully understood until today. Anthropogenic influences as submarine constructions, overfishing, eutrophication and climate warming are assumed as possible causes. Jellyfish and their relatives have fascinating capabilities and features, including their amazing regeneration potential, a high adaptability on changing environments and especially their weapons, the stinging capsules. Despite the simplicity of their nervous system jellyfish have astonishing perception senses.     

Production of G protein‐coupled receptors in an insect‐based cell‐free system

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein‐coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell‐free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell‐free system, performed within a short time and in a cost‐effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect‐based cell‐free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell‐free synthesized MOR in comparison to MOR expressed in a human cell line by “one‐point” radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328–2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Host plant genotype determines bottom‐up effects in an aphid‐parasitoid‐predator system

Plant genotypes are known to affect performance of insect herbivores and the community structure of both herbivores and higher trophic levels. Still, only a limited number of studies demonstrate differences in the performance of predators and parasitoids because of plant genotypic effects and most of these focus on gall formers. We designed a greenhouse experiment to investigate the effects of host plant genotype on fitness components in a grass‐aphid‐carnivore system. We used clones of quackgrass [Elytrigia repens (L.) Desv. ex Nevski (Poaceae)], the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae), the parasitoid wasp Aphidius colemani (Viereck) (Hymenoptera: Braconidae), and the predatory lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae). The number of aphid offspring differed considerably among plant genotypes. These differences were only in part because of differences in the production of biomass among host genotypes. Therefore, genotypes may differ in their nutritional value for phytophages. The number of aphids attacked by the parasitoid also differed among genotypes and aphid numbers only partly accounted for this effect. Moreover, pupal development time of female parasitoids was affected by plant genotype. We found no differences in mortality, body size, or sex ratio of hatching wasps between genotypes of quackgrass. Development time of the larvae and larval weight of the predatory lacewings differed among genotypes, but not weight of pupae and adults. Generally, the proportion of the total variance explained by the plant genotype was smaller for parasitoids and predators than for aphids. Overall, our experiments indicated that the plant genotype affects tri‐trophic interactions, but also that the strength of these effects decreases along the food chain.     

Co‐evolution‐driven switch of J‐protein specificity towards an Hsp70 partner

Molecular mechanisms by which protein–protein interactions are preserved or lost after gene duplication are not understood. Taking advantage of the well–studied yeast mtHsp70:J–protein molecular chaperone system, we considered whether changes in partner proteins accompanied specialization of gene duplicates. Here, we report that existence of the Hsp70 Ssq1, which arose by duplication of the gene encoding multifunction mtHsp70 and specializes in iron–sulphur cluster biogenesis, correlates with functional and structural changes in the J domain of its J–protein partner Jac1. All species encoding this shorter alternative version of the J domain share a common ancestry, suggesting that all short JAC1 proteins arose from a single deletion event. Construction of a variant that extended the length of the J domain of a ‘short’ Jac1 enhanced its ability to partner with multifunctional Hsp70. Our data provide a causal link between changes in the J protein partner and specialization of duplicate Hsp70.     

Tracking mesenchymal stromal cells using an ultra‐bright TAT‐functionalized plasmonic‐active nanoplatform

High‐resolution tracking of stem cells remains a challenging task. An ultra‐bright contrast agent with extended intracellular retention is suitable for in vivo high‐resolution tracking of stem cells following the implantation. Here, a plasmonic‐active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide‐functionalized gold nanostars (TAT‐GNS) that emit ultra‐bright two‐photon photoluminescence capable of tracking MSCs under high‐resolution optical imaging. In vitro experiment showed TAT‐GNS‐labeled MSCs retained a similar differentiability to that of non‐labeled MSCs controls. Due to their star shape, TAT‐GNS exhibited greater intracellular retention than that of commercial Q‐Tracker. In vivo imaging of TAT‐GNS‐labeled MSCs five days following intra‐arterial injections in mice kidneys showed possible MSCs implantation in juxta‐glomerular (JG) regions, but non‐specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic‐active nanoplatforms may be a useful intracellular tracking tool for stem cell research.

An ultra‐bright intracellular contrast agent is developed using TAT peptide‐functionalized gold nanostars (TAT‐GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two‐photon photoluminescence and superior labeling efficiency than commercial Q‐Tracker. Following renal implantation, some TAT‐GNS‐labeled MSCs permeate blood vessels and migrate to the juxta‐glomerular region.     

Leishmania donovani‐specific Ub‐related modifier‐1: an early endosome‐associated ubiquitin‐like conjugation in Leishmania donovani

Protein modification by ubiquitin (Ub) and Ub‐like molecules (Ubls) is a diverse biological process that regulates the activity of the target proteins. Systematic studies of Ubls in trypanosomatids like Leishmania, the causative organism of potentially fatal visceral leishmaniasis, would yield a better understanding of the disease pathogenesis and identify novel therapeutic targets. The present study is the first to characterize Leishmania donovani‐specific Ub‐related modifier‐1 (LdUrm1) and the associated conjugation pathway. Based on homology modeling, LdUrm1 was found to possess a β‐grasp fold and a C‐terminal di‐glycine motif unique to Ub/Ubls, essential for its conjugation to the target proteins. We identified LdUba4 as the E1 enzyme for LdUrm1 and demonstrated its energy‐dependent enzymatic activity. LdUrm1 was immunolocalized anteriorly near the flagellar reservoir, while LdUba4 was cytoplasmic, both in promastigotes and axenic amastigotes. Expression of nonconjugatable LdUrm1 in L. donovani resulted in depleted parasite growth suggesting its role in the pathogenesis. By mass spectrometry, we identified Rab5, a known mediator of early endosome regulated hemoglobin endocytosis in Leishmania, as a target of LdUrm1. Our data suggest that LdUrm1 conjugation pathway may have a role in early endosome‐mediated heme uptake in Leishmania that may be explored as a drug target.     

Generation of an estrogen receptor beta‐iCre knock‐in mouse

A novel knock‐in mouse that expresses codon‐improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatiotemporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the endogenous start codon of the Esr2 gene of a C57BL/6J embryonic stem cell line via homologous recombination. Resulting Esr2‐iCre mice were bred with ROSA26‐lacZ and Ai9‐RFP reporter mice to visualize cells of functional iCre expression. Strong expression was observed in the ovary, the pituitary, the interstitium of the testes, the head and tail but not body of the epididymis, skeletal muscle, the coagulation gland (anterior prostate), the lung, and the preputial gland. Additional diffuse or patchy expression was observed in the cerebrum, the hypothalamus, the heart, the adrenal gland, the colon, the bladder, and the pads of the paws. Overall, Esr2‐iCre mice will serve as a novel line for conditionally ablating genes in Esr2‐expressing tissues, identifying novel Esr2‐expressing cells, and differentiating the functions of ESR2 and ESR1. genesis 54:38–52, 2016. © 2016 Wiley Periodicals, Inc.