A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20^F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.     

In vitro activity of steroidal dendrimers on Trypanosoma cruzi epimastigote form with PAMAM dendrons modified by “click” chemistry

The increasing use of dendrimers shows promise for the treatment of inflammatory diseases, Chagas disease and other conditions such as cancer. In this study, the activity of 1st and 2nd generation dendrimers over T. cruzi in the epimastigote stage was tested. Dendrimers were derived from α-ethynylestradiol (EE) modified with PAMAM-type dendrons through a triazole ring. The activity of each compound was evaluated in five doses (from 1.3 to 20 µmol/mL) by flow cytometry, including benznidazole (Bz) as positive control. The findings show that an equivalent concentration of 14.8 µmol/mL of 2nd generation (G) dendrimer is 8 times more effective than Bz at 24 h, and it maintains its superiority at 48 h with an IC₅₀ = 1.25 ± 0.19 µmol/mL. A TUNEL assay showed that dendrimers induce cell death in T. cruzi epimastigotes mostly via apoptosis, unlike Bz, which induces death via necrosis in more than 50% of cells.     

Presence of a highly efficient “binding” to bacterial contamination can distort data from binding studies

[³H]GABA at low concentrations (5–10 nM) was bound by what appeared to be a GABA receptor binding site in bacterial contamination originating from a batch of distilled water. Under experimental conditions similar to those usually employed in [³H]GABA binding studies, the apparent binding displayed a very high specific component and a high efficiency in terms of [³H]GABA bound per mg of protein. The binding was blocked by muscimol but not by isoguvacine, SR95531 and nipecotic acid. These characteristics suggest that the presence of such spurious binding in the experiments using³H-labeled ligands in brain homogenates may not always be very obvious and, morover, it can result in subtle, but serious, distortions of data from such studies, which may not be immediately recognized.     

Fixed-bed dialysis culture of a transfectoma cell line producing chimeric Fab-fragments with “nutrient-split”-feeding strategy

The very shear sensitive transfectoma cell line, FAB 10, producing a chimeric Fab-antibody fragment specific for the human carcinoembryonic antigen (CEA), was cultivated in fixed bed reactors where the cells were immobilized in macroporous carriers. As the cell line had a very low productivity, cultures with process integrated product enrichment were performed in a membrane dialysis bioreactor with integrated fixed bed, where low molecular weight metabolites are removed over the membrane and high molecular weight products are enriched. In a new nutrient-split feeding strategy for dialysis cultures concentrated medium was supplied directly to the fixed bed unit, whereas a buffer solution was used as dialysis fluid. With the dialysis technique up to 5800 g Fab l–1 could be obtained, more than 10 times higher compared to fixed bed cultures without dialysis or batch cultures with suspended cells. The nutrient-split-feeding strategy reduced the amount of medium required per mg of antibody significantly. © Rapid Science Ltd. 1998     

“Use of acidophilic bacteria of the genus Acidithiobacillus to biosynthesize CdS fluorescent nanoparticles (quantum dots) with high tolerance to acidic pH”

The use of bacterial cells to produce fluorescent semiconductor nanoparticles (quantum dots, QDs) represents a green alternative with promising economic potential. In the present work, we report for the first time the biosynthesis of CdS QDs by acidophilic bacteria of the Acidithiobacillus genus. CdS QDs were obtained by exposing A. ferrooxidans, A. thiooxidans and A. caldus cells to sublethal Cd²⁺ concentrations in the presence of cysteine and glutathione. The fluorescence of cadmium-exposed cells moves from green to red with incubation time, a characteristic property of QDs associated with nanocrystals growth. Biosynthesized nanoparticles (NPs) display an absorption peak at 360 nm and a broad emission spectra between 450 and 650 nm when excited at 370 nm, both characteristic of CdS QDs. Average sizes of 6 and 10 nm were determined for green and red NPs, respectively. The importance of cysteine and glutathione on QDs biosynthesis in Acidithiobacillus was related with the generation of H₂S. Interestingly, QDs produced by acidophilic bacteria display high tolerance to acidic pH. Absorbance and fluorescence properties of QDs was not affected at pH 2.0, a condition that totally inhibits the fluorescence of QDs produced chemically or biosynthesized by mesophilic bacteria (stable until pH 4.5–5.0). Results presented here constitute the first report of the generation of QDs with improved properties by using extremophile microorganisms.     

A Function Essential to Viral Entry Underlies the Hepatitis B Virus “a” Determinant

The hepatitis B virus (HBV) particles bear a receptor-binding site located in the pre-S1 domain of the large HBV envelope protein. Using the hepatitis delta virus (HDV) as a surrogate of HBV, a second infectivity determinant was recently identified in the envelope proteins antigenic loop (AGL), and its activity was shown to depend upon cysteine residues that are essential for the structure of the HBV immunodominant “a” determinant. Here, an alanine-scanning mutagenesis approach was used to precisely map the AGL infectivity determinant to a set of conserved residues, which are predicted to cluster together with cysteines in the AGL disulfide bridges network. Several substitutions suppressed both infectivity and the “a” determinant, whereas others were infectivity deficient with only a partial impact on antigenicity. Interestingly, G145R, a substitution often arising under immune pressure selection and detrimental to the “a” determinant, had no effect on infectivity. Altogether, these findings indicate that the AGL infectivity determinant is closely related to, yet separable from, the “a” determinant. Finally, a selection of HDV entry-deficient mutations were introduced at the surface of HBV virions and shown to also abrogate infection in the HBV model. Therefore, a function can at last be assigned to the orphan “a” determinant, the first-discovered marker of HBV infection. The characterization of the AGL functions at viral entry may lead to novel approaches in the development of antivirals against HBV.Hepatitis B virus (HBV) causes acute and chronic infections in humans; such infections are often associated with severe liver diseases, including cirrhosis and hepatocellular carcinoma (10). To date, it is estimated that approximately 350 millions individuals worldwide suffer from chronic infection despite the availability of an effective vaccine for more than 25 years. Remarkably, the development of a vaccine soon after the HBV discovery was, at least in part, the consequence of a very peculiar feature that is unique to members of the Hepadnaviridae family: viral envelope proteins are produced in quantities far exceeding the amounts required for assembly of HBV virions (6) and, owing to their capacity for autoassembly, the vast majority are secreted as subviral particles. Besides the practical consequences in the original vaccine development, in nature, the phenomenon of HBV envelope protein overexpression has provided a helper function to the hepatitis delta virus (HDV) (29). The HBV envelope proteins assist in packaging the HDV ribonucleoprotein (RNP) in case of HBV-HDV coinfection, thereby ensuring spreading of the satellite HDV. As a result, the coats of HBV and HDV particles are similar, consisting of cell-derived lipids and the HBV envelope proteins—large, middle, and small—bearing the HBV surface antigen (HBsAg) and referred to as L-HBsAg, M-HBsAg, and S-HBsAg, respectively (4, 14).The HBsAg includes an immunodominant determinant common to all HBV strains, referred to as “a,” and several mutually exclusive subtype-specific determinants referred to as “d”/“y” and “w”/“r” (21). The “a” determinant is defined by a specific conformation of the antigenic loop (AGL) polypeptide present at the surface of subviral, HBV, or HDV particles. The AGL itself resides between the transmembrane domain II (residues 80 to 100) and the hydrophobic carboxyl terminus (residues 165 to 226) of the envelope proteins S domain (see Fig. ​Fig.1).1). It is the “a” determinant that elicits the most effective neutralizing antibody response upon vaccination or infection (32). Surprisingly, a function in the HBV life cycle had never been assigned to the “a” determinant, the first identified HBV marker, until the recent demonstration of its involvement in HDV entry (2, 15). More precisely, it was shown that the AGL cysteine residues were critical for both the structure of the “a” determinant and HDV infectivity (2).Open in a separate windowFIG. 1.Schematic representation of the HBV envelope protein AGL. (A) The topology of the L-, M-, and S-HBsAg proteins (L, M, and S, respectively) is represented. The determinants of viral entry, pre-S1 and AGL, are indicated in red. The M-HBsAg protein, represented in gray, is dispensable for infectivity. Open boxes represent transmembrane regions in the S domain. (B) Alignment of the AGL amino acids sequences (positions 101 to 172 in the S domain) of HBV (genotype D, ayw3 phenotype), WMHBV, and WHV. The GenBank sequence numbers of the isolates are as follows: {"type":"entrez-nucleotide","attrs":{"text":"J02203","term_id":"329640","term_text":"J02203"}}J02203 (HBV), {"type":"entrez-nucleotide","attrs":{"text":"AY226578","term_id":"29501369","term_text":"AY226578"}}AY226578 (WMHBV), and {"type":"entrez-nucleotide","attrs":{"text":"NC_004107","term_id":"22256030","term_text":"NC_004107"}}NC_004107 (WHV). HBV amino acid residues important for infectivity (the present study) are indicated in blue. A hyphen denotes amino acid identity with the HBV sequence.It is now well established that both HBV and HDV entry rely on the pre-S1 domain of L-HBsAg as the primary infectivity determinant that is likely to promote attachment to a specific receptor at the surface of human hepatocytes (11). The AGL determinant could thus fulfill complementary functions for attachment, uptake, or particle disassembly after entry (2, 15).In the present study, the AGL infectivity determinant was mapped and confirmed to be closely related to the “a” determinant. Moreover, its essential function at viral entry was demonstrated in the HBV model.     

“Just Get on with It”: Qualitative Insights of Coming to Terms with a Deteriorating Body for Older Women with Osteoarthritis

Objective

To qualify the psychosocial burden of osteoarthritis for older women and identify factors perceived to assist with psychological adjustment to the disease.

Methods

Women who indicated being diagnosed/treated for osteoarthritis in the previous three years in the fifth survey of the Australian Longitudinal Study on Women’s Health provided the sampling frame. Participants were randomly sampled until saturation was reached using a systematic process. Thematic content analysis was applied to the 19 semi-structured telephone interviews using a realist framework.

Results

The findings indicate that the emotional burden of osteoarthritis is considerable, and the process of psychological adjustment complex. Older women with osteoarthritis have psychological difficulties associated with increasing pain and functional impairment. Psychological adjustment over time was attributed primarily to cognitive and attitudinal factors (e.g. stoicism, making downward comparisons and possessing specific notions about the cause of arthritis). This was a dynamic ‘day to day’ process involving a constant struggle between grieving physical losses and increasing dependence amidst symptom management.

Conclusion

The findings of this study add to the current understanding of the complex processes involved in psychological adjustment over time. Targeted interventions focused on assisting women with arthritis redefine self-concepts outside the confines of caring responsibilities, coupled with public health education programs around understanding the destructive nature of arthritis are required. Understanding the destructive and (potentially) preventable nature of arthritis may facilitate early detection and increased uptake of appropriate treatment options for osteoarthritis that have the ability to modify disease trajectories.     

Coreceptor gene “imprinting:” A genetic solution to a developmental dilemma in T cells

Comment on: Adoro S, et al. EMBO J 2011; 31:366-77.     

Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of “heat-controlled necrosis” with heat shock protein expression

Heat shock proteins (HSPs) are highly conserved proteins whose syntheses are induced by a variety of stresses, including heat stress. Since the expression of HSPs, including HSP70, protects cells from heat-induced apoptosis, HSP expression has been considered to be a complicating factor in hyperthermia. On the other hand, recent reports have shown the importance of HSPs, such as HSP70, HSP90 and glucose-regulated protein 96 (gp96), in immune reactions. If HSP expression induced by hyperthermia is involved in tumor immunity, novel cancer immunotherapy based on this novel concept can be developed. In such a strategy, a tumor-specific hyperthermia system, which can heat the local tumor region to the intended temperature without damaging normal tissue, would be highly advantageous. To achieve tumor-specific hyperthermia, we have developed an intracellular hyperthermia system using magnetite nanoparticles. This novel hyperthermia system can induce necrotic cell death via HSP expression, which induces antitumor immunity. In the present article, cancer immunology and immunotherapy based on hyperthermia, and HSP expression are reviewed and discussed. This article forms part of the Symposium in Writing "Thermal stress-related modulation of tumor cell physiology and immune responses", edited by Elfriede Noessner.     

Why Was Thomas A. Sebeok Not a Cognitive Ethologist? From “Animal Mind” to “Semiotic Self”

In the current debates about zoosemiotics its relations with the neighbouring disciplines are a relevant topic. The present article aims to analyse the complex relations between zoosemiotics and cognitive ethology with special attention to their establishers: Thomas A. Sebeok and Donald R. Griffin. It is argued that zoosemiotics and cognitive ethology have common roots in comparative studies of animal communication in the early 1960s. For supporting this claim Sebeok’s works are analysed, the classical and philosophical periods of his zoosemiotic views are distinguished and the changing relations between zoosemiotics and cognitive ethology are described. The animal language controversy can be interpreted as the explicit point of divergence of the two paradigms, which, however, is a mere symptom of a deeper cleavage. The analysis brings out later critical differences between Sebeok’s and Griffin’s views on animal cognition and language. This disagreement has been the main reason for the critical reception and later neglect of Sebeok’s works in cognitive ethology. Sebeok’s position in this debate remains, however, paradigmatic, i.e. it proceeds from understanding of the contextualisation of semiotic processes that do not allow treating the animal mind as a distinct entity. As a peculiar parallel to Griffin’s metaphor of “animal mind”, Sebeok develops his understanding of “semiotic self” as a layered structure, characterised by an ability to make distinctions, foremost between itself and the surrounding environment. It appears that the history of zoosemiotics has two layers: in addition to the chronological history starting in 1963, when Sebeok proposed a name for the field, zoosemiotics is also philosophically rooted in Peircean semiotics and German biological philosophy. It is argued that the confrontation between zoosemiotics and cognitive ethology is related to different epistemological approaches and at least partly induced by underlying philosophical traditions.     

The pepino(Solanum muricatum,Solanaceae): A “New” crop with a history

Pepino,Solanum muricatum, is an herbaceous subshrub that has long been grown in its native Andean South America. Pepino is usually cultivated for its edible fruits, but also has other economic uses. In spite of being a prominent crop in prehispanic times in the Andes, interest in pepino was cast into oblivion from some decades after the Spanish arrival to the present. Pepino etymology, prehispanic distribution, and postcolumbian dispersal are presented, with emphasis on outstanding historical aspects. Speculations on why the pepino has been neglected are also given. These include some features of pepino itself together with misconceptions. However, the pepino is today a species of increasing economic interest, and has a considerable potential for future exploitation.     

Estimation of signal and noise spectra by special averaging techniques with application to a posteriori “Wiener” filtering

This paper deals with the problem of separating the spectra of signal and noise in ensembles where the signal can be considered as an invariant component and the noise as a stationary additive background. Several methods are discussed and compared on the basis of a statistical analysis of the first two moments of the estimators for signal and noise spectra. As a consequence a procedure is proposed which provides a flexible compromise between estimation accuracy and computational effort. The application of this procedure to a posteriori Wiener filtering is compared with a more common, but time consuming, technique.     

A “Bat” Is Easier to Learn than a “Tab”: Effects of Relative Phonotactic Frequency on Infant Word Learning

Many studies have shown that during the first year of life infants start learning the prosodic, phonetic and phonotactic properties of their native language. In parallel, infants start associating sound sequences with semantic representations. However, the question of how these two processes interact remains largely unknown. The current study explores whether (and when) the relative phonotactic probability of a sound sequence in the native language has an impact on infants’ word learning. We exploit the fact that Labial-Coronal (LC) words are more frequent than Coronal-Labial (CL) words in French, and that French-learning infants prefer LC over CL sequences at 10 months of age, to explore the possibility that LC structures might be learned more easily and thus at an earlier age than CL structures. Eye movements of French-learning 14- and 16-month-olds were recorded while they watched animated cartoons in a word learning task. The experiment involved four trials testing LC sequences and four trials testing CL sequences. Our data reveal that 16-month-olds were able to learn the LC and CL words, while14-month-olds were only able to learn the LC words, which are the words with the more frequent phonotactic pattern. The present results provide evidence that infants’ knowledge of their native language phonotactic patterns influences their word learning: Words with a frequent phonotactic structure could be acquired at an earlier age than those with a lower probability. Developmental changes are discussed and integrated with previous findings.     

Facultative nest patch shifts in response to nest predation risk in the Brewer’s sparrow: a “win-stay, lose-switch” strategy?

Facultative shifts in nesting habitat selection in response to perceived predation risk may allow animals to increase the survival probability of sessile offspring. Previous studies on this behavioral strategy have primarily focused on single attributes, such as the distance moved or changes in nesting substrate. However, nest site choice often encompasses multiple habitat elements at both the nest site and nest patch scales. We studied the within-season re-nesting strategy of a multi-brooded songbird, the Brewer’s sparrow (Spizella breweri), to determine whether pairs utilized a “win-stay, lose-switch” decision rule with respect to inter-nest distance, nest substrate and/or nest patch characteristics in response to previous nest fate. Pairs moved sequential nest sites slightly farther following nest predation versus success. When inter-nest distance was controlled, however, pairs changed nest patch attributes (shrub height, potential nest shrub density) associated with probability of nest predation to a greater extent following nest predation than success. The strategy appeared to be adaptive; daily nest survival probability for previously depredated pairs increased with greater Euclidian habitat distances between attempts, whereas previously successful pairs were more likely to fledge second attempts when nest sites were similar to those of previous attempts. Our results suggest that nesting birds can use prior information and within-season plasticity in response to nest predation to increase re-nesting success, which may be a critical behavioral strategy within complex nest predator environments. Re-nesting site selection strategies also appeared to integrate multiple habitat components and inter-nest distances. The consideration of such proximate, facultative responses to predation risk may clarify often unexplained variation in habitat preferences and requirements.