Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.     

Visual Cues Given by Humans Are Not Sufficient for Asian Elephants (Elephas maximus) to Find Hidden Food

Recent research suggests that domesticated species – due to artificial selection by humans for specific, preferred behavioral traits – are better than wild animals at responding to visual cues given by humans about the location of hidden food. \Although this seems to be supported by studies on a range of domesticated (including dogs, goats and horses) and wild (including wolves and chimpanzees) animals, there is also evidence that exposure to humans positively influences the ability of both wild and domesticated animals to follow these same cues. Here, we test the performance of Asian elephants (Elephas maximus) on an object choice task that provides them with visual-only cues given by humans about the location of hidden food. Captive elephants are interesting candidates for investigating how both domestication and human exposure may impact cue-following as they represent a non-domesticated species with almost constant human interaction. As a group, the elephants (n = 7) in our study were unable to follow pointing, body orientation or a combination of both as honest signals of food location. They were, however, able to follow vocal commands with which they were already familiar in a novel context, suggesting the elephants are able to follow cues if they are sufficiently salient. Although the elephants’ inability to follow the visual cues provides partial support for the domestication hypothesis, an alternative explanation is that elephants may rely more heavily on other sensory modalities, specifically olfaction and audition. Further research will be needed to rule out this alternative explanation.     

Transition metal and organic radical components of carp liver tissue observed by electron paramagnetic resonance spectroscopy

Paramagnetic transition metal centers and organic radicals in liver from wild-type carp (Cyprinus carpio) were characterized by electron paramagnetic resonance (EPR) spectroscopy. Approximately twelve EPR signals were observed at 77 K with resonance positions between g=1.8 and g=2.5. Identification was facilitated by a study of the variation in signal intensity with microwave power (microwave power saturation) for each signal. Many were organic radical or iron signals from typical liver enzymes, including cytochrome P450, coenzyme Q₁₀, NADH dehydrogenase, and succinate dehydrogenase, cytochrome c oxidase and/or catalase. Of special interest were two signals that are not normally found in mammalian liver. The first was a six-line signal from divalent manganese, which was evident in the spectra in quantities suggestive of a functional role. The second was probably a signal from nitrosylated non-heme iron and may be related to the presence of nitrogen-containing compounds produced by nitrifying bacteria in the aquatic environment. These notable differences between the EPR spectra of fish and mammalian liver suggest major metabolic differences between the two systems.     

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.     

A phytochemical study of the quinolizidine alkaloids from Genista tenera by gas chromatography-mass spectrometry

Gas chromatography coupled with mass spectrometry has been used to analyse the alkaloids present in the aerial parts of Genista tenera. Anagyrine, cytisine, N-formylcytisine, N-methylcytisine and lupanine were the major compounds, the last two alkaloids being known for their hypoglycaemic activity. Dehydrocytisine, 5,6-dehydrolupanine, rhombifoline, aphylline and thermopsine were the minor alkaloids. The characterisation of the constituents was based on comparison of their Kovats retention indexes and electron impact-mass spectrometric data recorded on-line with those of reference compounds and literature data.     

Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the beta-defensin family, human beta-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of beta-defensins is mediated by at least three different mechanisms.     

Comprehensive phylogeny of the laughingthrushes and allies (Aves,Leiothrichidae) and a proposal for a revised taxonomy

DNA phylogenies have gradually shed light on the phylogenetic relationships of the large babbler group. We focus in this study on the family Leiothrichidae (laughingthrushes and “song babblers”), which represents the largest clade of babblers in terms of species diversity. Our phylogeny includes all genera and 82% of the recognized species, using mitochondrial and nuclear loci. The sister group to Leiothrichidae is composed of the Pellorneidae (“jungle babblers”) plus the genus Alcippe. Within Leiothrichidae, four strongly supported primary clades (A–D) are recovered. Clade A includes Grammatoptila, Laniellus and Cutia. Clade B includes a large group of laughingthrushes, all of them classified in Trochalopteron. In Clade C, the two laughingthrushes endemic to southern India, T. fairbanki and T. cachinnans, which have recently been proposed to be placed in the newly erected genus Montecincla, form a sister clade to the group comprising the “song babblers” (Lioptila, Leiothrix, Heterophasia, Minla, Liocichla, Actinodura, Chrysominla, Siva, and Sibia). Clade D includes the African babblers (Turdoides, Phyllanthus, Kupeornis), Asian relatives (Argya, Acanthoptila, Chatarrhaea) and all remaining laughingthrushes (Garrulax). The time estimates suggest that the early diversification of the Leiothrichidae occurred in the mid‐Miocene, a period that corresponds to the diversification of many passerine groups in Asia. A revised taxonomic classification of the family is proposed in the light of these results.     

Retinol (Vitamin A) Increases α-Synuclein, β-Amyloid Peptide,Tau Phosphorylation and RAGE Content in Human SH-SY5Y Neuronal Cell Line

Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson’s disease and Alzheimer’s disease: α-synuclein, β-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, β-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and β-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.