Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst

Yeast Surface Display (YSD) is a strategy to anchor proteins on the yeast cell wall which has been employed to increase enzyme stability thus decreasing production costs. Lipase B from Candida antarctica (LipB) is one of the most studied enzymes in the context of industrial biotechnology. This study aimed to assess the biochemical features of this important biocatalyst when immobilized on the cell surface of the methylotrophic yeast Pichia pastoris using the YSD approach. For that purpose, two anchors were tested. The first (Flo9) was identified after a prospection of the P. pastoris genome being related to the family of flocculins similar to Flo1 but significantly smaller. The second is the Protein with Internal Repeats (Pir1) from P. pastoris. An immunolocalization assay showed that both anchor proteins were able to display the reporter protein EGFP in the yeast outer cell wall. LipB was expressed in P. pastoris fused either to Flo9 (FLOLIPB) or Pir1 (PIRLIPB). Both constructions showed hydrolytic activity towards tributyrin (>100 U/mg_dcw and >80 U/mg_dcw, respectively), optimal hydrolytic activity around 45°C and pH 7.0, higher thermostability at 45°C and stability in organic solvents when compared to a free lipase.     

Human polyreactive and monoreactive antibodies: effect of glycosylation antigen binding

The present experiments were initiated to determine whetherthe carbohydrate portions of antibody molecules contribute topolyreactivity. Cell lines making human monoclonal polyreactiveor monoreactive antibodies of the immunoglobulin (Ig) M, IgGand IgA isotypes were treated with tunicamycin to block N-linkedglycosylation of the proteins. Analysis of the secreted nativeand non-glycosylated proteins revealed a >95% inhibitionof [³H]mannose incorporation. Electrophoresis on sodium dodecylsulphate-polyacrylamide gels of the proteins from tunicamycin-treatedcells showed increased mobility and the absence of [³H]mannoseincorporation of the immunoglobulin heavy chains, consistentwith the lack of glycosylation. The native and non-glycosylatedantibodies were then tested for their ability to bind differentantigens. Despite the lack of glycosylation, both polyreactiveand monoreactive antibodies bound to antigens with little ifany loss of reactivity or specificity. It is concluded thatthe carbohydrate moieties do not contribute significantly topolyreactivity. antigen binding glycosylation polyreactive antibodies     

Ectogenesis and gender-based oppression: Resisting the ideal of assimilation

In a recent article in this journal, Kathryn MacKay advances a defence of ectogenesis that is grounded in this technology’s potential to end—or at least mitigate the effects of—gender-based oppression. MacKay raises important issues concerning the socialization of women as ‘mothers’, and the harms that this socialization causes. She also considers ectogenesis as an ethically preferable alternative to gestational surrogacy and uterine transplantation, one that is less harmful to women and less subject to being co-opted to further oppressive ends. In this article, I challenge some of the assumptions that underlie MacKay’s case in favour of ectogenesis by questioning whether the relationship between women’s capacity to gestate and birth children and gender-based oppression is as strong as MacKay makes it out to be. I subsequently argue that—even if MacKay’s reading of this relationship is accurate—ectogenesis is not a desirable means to end gender-based oppression. It embodies a strategy that could be used to pursue liberating projects that follow what Iris Marion Young defines as ‘the ideal of assimilation’, but that must be resisted. I then concur with MacKay’s contention that ectogenesis is better than gestational surrogacy and uterine transplantation. My argument is that many of the problematic issues that MacKay herself sees as features of these practices will not disappear with ectogenesis. Finally, I conclude that MacKay’s narrow focus on women’s biology and ectogenesis as a solution to gender-based oppression results in the overlooking of broader systemic issues that contribute to the upholding of oppressive norms.     

Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase

The enzymes phosphomannomutase (PMM), phospho‐N‐acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose, N‐acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome, there is, surprisingly, no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Å resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite, while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose‐phosphates, as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that, in vivo, PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why, uniquely, T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.     

Effects of assessing the productivity of faculty in academic medical centres: a systematic review

Background:

Many academic medical centres have introduced strategies to assess the productivity of faculty as part of compensation schemes. We conducted a systematic review of the effects of such strategies on faculty productivity.

Methods:

We searched the MEDLINE, Healthstar, Embase and PsycInfo databases from their date of inception up to October 2011. We included studies that assessed academic productivity in clinical, research, teaching and administrative activities, as well as compensation, promotion processes and satisfaction.

Results:

Of 531 full-text articles assessed for eligibility, we included 9 articles reporting on eight studies. The introduction of strategies for assessing academic productivity as part of compensation schemes resulted in increases in clinical productivity (in six of six studies) in terms of clinical revenue, the work component of relative-value units (these units are nonmonetary standard units of measure used to indicate the value of services provided), patient satisfaction and other departmentally used standards. Increases in research productivity were noted (in five of six studies) in terms of funding and publications. There was no change in teaching productivity (in two of five studies) in terms of educational output. Such strategies also resulted in increases in compensation at both individual and group levels (in three studies), with two studies reporting a change in distribution of compensation in favour of junior faculty. None of the studies assessed effects on administrative productivity or promotion processes. The overall quality of evidence was low.

Interpretation:

Strategies introduced to assess productivity as part of a compensation scheme appeared to improve productivity in research activities and possibly improved clinical productivity, but they had no effect in the area of teaching. Compensation increased at both group and individual levels, particularly among junior faculty. Higher quality evidence about the benefits and harms of such assessment strategies is needed.Academic productivity can be defined as a measurable output of a faculty member related to clinical, research, education or administrative activities. Achieving the best possible academic productivity is essential for academic medical centres to maintain or nurture recognition and good reputation.¹ Furthermore, clinical productivity is essential for the survival of academic departments given the economic realities in medicine.²Strategies for productivity assessment help in identifying highly productive faculty, determining areas for faculty and departmental improvement,³ and implementing processes for promotion and tenure.⁴ When coupled with reward schemes, these strategies may improve productivity and compensation at both individual and departmental levels. In the long-term, they may enhance the ability to recruit and retain high-quality faculty and achieve the academic mission of the department. However, these strategies may have some unintended effects such as using time dedicated to education to do more clinical work.³ In addition, they may be challenging to implement.³We conducted a systematic review of the effects of strategies introduced in academic medical centres to assess faculty productivity, compensation, promotion processes and satisfaction.     

Interpopulational variation of the mating system in the peacock blenny Salaria pavo

The ecology and mating system of two populations of the peacock blenny Salaria pavo that have been reported to differ in sexual behaviour were studied. In the Gulf of Trieste, a northern Adriatic Sea population lives in rocky shores, whilst in the Ria Formosa, an Atlantic population inhabits a coastal lagoon with sandy barrier islands. In the Gulf of Trieste, nest availability was found to be higher and nest aggregation lower than in Ria Formosa. Nesting males courted females more and in higher proportion, and inversely, females directed less courtship displays towards nest holders in the Gulf of Trieste than in Ria Formosa. The relative frequency of small female-like males that reproduce by parasitically fertilizing eggs in the larger males’ nests was lower in the Gulf of Trieste population. This variation in the sexual behaviour of nesting males and females is likely to reflect a plastic behavioural response to the varying ecological conditions. The difference between populations in the relative frequency of parasitic males suggests that the male alternative reproductive tactics are condition-dependent.     

7-Substituted-pyrrolo[3,2-d]pyrimidine-2,4-dione derivatives as antagonists of the transient receptor potential ankyrin 1 (TRPA1) channel: a promising approach for treating pain and inflammation

The transient receptor potential ankyrin 1 (TRPA1) channel is activated by a series of by-products of oxidative/nitrative stress, produced under inflammatory conditions or in the case of tissue damage, thus generating inflammatory and neuropathic pain and neurogenic inflammatory responses. These findings have identified TRPA1 as an emerging opportunity for the design and synthesis of selective inhibitors as potential analgesic and antiinflammatory agents. Herein we present the synthesis and functional evaluation of a new series of 7-substituted-1,3-dimethyl-1,5-dihydro-pyrrolo[3,2-d]pyrimidine-2,4-dione derivatives designed as TRPA1 antagonists. A small library of compounds has been built by the introduction of differently substituted N(7)-phenylacetamide or N(7)-[4-(substituted-phenyl)-thiazol-2-yl]-acetamide chains. All the synthesized compounds were assayed to evaluate their ability to block acrolein-mediated activation of native human and rat TRPA1 channels employing a fluorometric calcium imaging assay. Our study led us to the identification of compound 3h which showed considerably improved potency (IC(50)=400nM) against human TRPA1 with regard to some of the most representative antagonists previously reported and integrated in our screening program as reference compounds. In addition, 3h proved to maintain its efficacy toward rTRPA1, which designates it as a possible candidate for future evaluation of in vivo efficacy in rodent animal model of inflammatory and neuropathic pain.