High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg?l^?1 of enzyme with a biomass yield of 16.2 mg?g_cdw ^?1 and a productivity of 10.5 mg?l^?1?h^?1.     

Biofilms: the environmental playground of Legionella pneumophila

Legionella pneumophila, the aetiological agent of 90% of legionellosis cases, is a common inhabitant of natural and anthropogenic freshwater environments, where it resides in biofilms. Biofilms are defined as complex, natural assemblages of microorganisms that involve a multitude of trophic interactions. A thorough knowledge and understanding of Legionella ecology in relation to biofilm communities is of primary importance in the search for innovative and effective control strategies to prevent the occurrence of disease cases. This review provides a critical update on the state‐of‐the‐art progress in understanding the mechanisms and factors affecting the biofilm life cycle of L. pneumophila. Particular emphasis is given to discussing the different strategies this human pathogen uses to grow and retain itself in biofilm communities. Biofilms develop not only at solid‐water interfaces (substrate‐associated biofilms), but also at the water‐air interface (floating biofilms). Disturbance of the water surface can lead to liberation of aerosols derived from the floating biofilm into the atmosphere that allow transmission of biofilm‐associated pathogens over considerable distances. Recent data concerning the occurrence and replication of L. pneumophila in floating biofilms are also elaborated and discussed.     

Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(?)< 5 min) than the wild-type enzyme (t(?)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(?)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.     

Natural pathology of the captive chimpanzee (Pan troglodytes): A 35‐year review

We present the spontaneous pathological lesions identified as a result of necropsy or biopsy for 245 chimpanzees (Pan troglodytes) over a 35‐year period. A review of the pathology database was performed for all diagnoses on chimpanzees from 1980 to 2014. All morphologic diagnoses, associated system, organ, etiology, and demographic information were reviewed and analyzed. Cardiomyopathy was the most frequent lesion observed followed by hemosiderosis, hyperplasia, nematodiasis, edema, and hemorrhage. The most frequently affected systems were the gastrointestinal, cardiovascular, urogenital, respiratory, and lymphatic/hematopoietic systems. The most common etiology was undetermined, followed by degenerative, physiologic, neoplastic, parasitic, and bacterial. Perinatal and infant animals were mostly affected by physiologic etiologies and chimpanzee‐induced trauma. Bacterial and physiologic etiologies were more common in juvenile animals. Degenerative and physiologic (and neoplastic in geriatric animals) etiologies predominated in adult, middle aged, and geriatric chimpanzees.     

Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice

Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.     

Programmed anuclear cell death delimits platelet life span

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.     

PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.     

Male tree weta are attracted to cuticular scent cues but do not discriminate according to sex or among two closely related species

Recognition of conspecifics is an essential precursor of successful mating. Where related species coexist, species discrimination might be important, but because related species are similar, species signal recognition may actually be low. Chemical cues such as cuticular hydrocarbons (CHCs) are frequently used by insects to identify suitable sexual partners. We predicted that New Zealand tree weta (Hemideina spp.), a genus of nocturnal ensiferan Orthoptera that live both allopatrically and sympatrically, use chemical signals from either frass or CHCs to find mates. In a series of six laboratory trials using both H. thoracica and H. crassidens, we found that male tree weta, but not female tree weta, occupied cavities primed with female cuticular cues more often than cavities without. However, males did not discriminate between chemical cues of male and female conspecifics, or between conspecifics and heterospecifics. In field trials, tree weta did not occupy artificial cavities primed with either female frass or female cuticular cues more often than unscented cavities. However, in both trials weta preferentially returned to cavities that had already been occupied earlier in the trials. A final field trial confirmed the presence of mixed species harems during the mating season in one region of sympatry. Our results suggest that selection on sex and species specific chemical cues that could be used to find conspecific mates is weak. Mixed species aggregations suggest that identification of conspecific mating cues has not evolved to be species specific. We infer that for male tree weta, the cost of mating with heterospecifics is likely less than not mating at all.