Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rond?nia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.     

Developmental regulation of an instability element from the Drosophila fushi tarazu mRNA

Summary: The Drosophila fushi tarazu (ftz) mRNA is one of the shortest‐lived metazoan mRNAs, and its instability is crucial for proper development of the embryo. Previously, we identified two cis‐acting elements that are required for ftz mRNA degradation, one within the 5′ one‐third and another in the 3′UTR of the message. Here we focus on the 3′UTR element termed FIE3 (ftz instability e lement in the 3 ′UTR). To investigate the developmental regulation of the FIE3‐dependent degrading activity we measured the abundance of an FIE3‐containing mRNA in ovaries, unfertilized eggs, and different larval and adult tissues. We found that FIE3‐degrading activity is present at all developmental stages and tissues examined, except in the ovary. Activation of the FIE3‐dependent mRNA decay is independent of fertilization because it could be triggered by egg activation. Finally, we provide evidence that mutation of conserved elements within FIE3 had no effect on mRNA instability. genesis 30:59–64, 2001. © 2001 Wiley‐Liss, Inc.     

Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding

The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.     

Product social impact assessment

Purpose

Whereas the business evolution of environmental sustainability metrics has advanced significantly over the past decade, social sustainability at product level is still relatively immature. Research continues to support the front runners on organisational sustainability, while workable solutions at product level have not yet been addressed sufficiently. Triggered by this imbalance, a group of experts from large companies decided to join forces, initiating the Roundtable for Product Social Metrics.

Methods

Starting in early 2013, this group of companies aimed to (i) consolidate principles for product social sustainability assessment and harmonise approaches, (ii) align with other global initiatives and share with other companies and (iii) develop solutions for cross-cutting implementation issues. In order to be able to produce a comprehensive method for social impact assessment that provides enough flexibility for individual requirements, the Roundtable developed a method based on the approaches of the participant companies and external references such as the UNEP/SETAC Guidelines for Social Life-Cycle Assessment of Products and corporate level standards. Guiding principles were defined for the development of the method.

Results and discussion

The results of the first two phases of the Roundtable for Product Social Metrics are documented in a handbook, which proposes a practical method for organisations to assess the social impacts of a product or a service along its life cycle. The handbook outlines an aligned method for social impact assessment at a product level offering two approaches: quantitative and scale based. The method was developed to allow reasoned assessment of overall performance by including social topics and performance indicators that reflect positive and negative impacts of the product on three stakeholder groups: workers, consumers and local communities. Nineteen social topics are proposed, together with their individual performance indicators, including detailed definitions. Application examples and recommendations for the communication of results are also included in the handbook.

Conclusions

The method can be applied in numerous scenarios, from understanding improvement opportunities and steering product development in different stages, to providing support for decision making and external communications. However, the method still has further potential for improvement, inter alia that the proposed indicators are not fully applicable to small farmers, SMEs and the self-employed, as well as that the indicators are mainly at inventory level. Furthermore, the proposed method is strongly dependent on the availability of data.

     

Towards social life cycle assessment: a quantitative product social impact assessment

Purpose

The main goal of this paper is to present the feasibility of the quantitative method presented in the Product Social Impact Assessment (PSIA) handbook throughout a case study. The case study was developed to assess the social impacts of a tire throughout its entire life cycle. We carried out this case study in the context of the Roundtable for the Product Social Metrics project in which 13 companies develop two methodologies, a qualitative and a quantitative one, for assessing the social impact of product life cycle.

Methods

The quantitative methodology implemented for assessing the social impact of a Run On Flat tire mounted in a BMW 3 series consists of 26 indicators split in three groups. Each group represents a stakeholder group. Primary data of the quantitative indicators were collected along the product life cycle of the Run On Flat by involving the companies, which owned the main steps of the product life cycle. Throughout this case study, an ideal/worst-case scenario was defined for the distance-to-target approach to compare the social performances of more products when they are available.

Results and discussion

The implementation of the PSIA quantitative method to a Run On Flat illustrated the necessity to have a referencing step in order to interpret the results. This is particularly important when the results are used to support decision-making process in which no experts are involved. It frequently happens in a big company where the management level has to take often decisions on different topics. Reference values were defined using ideal or worst-case-target scenarios (Fontes et al. 2014). For those topics where it was possible, an ideal/ethical scenario was defined, e.g., 0 h of child labor per product. In other cases, we defined a worst-case scenario, e.g., 0 training hours per product. It was then possible to interpret the results using a distance-to-target approach. A matrix was developed in the case study for identifying in which step of the product life cycle data is not available; that means we need more transparency in the supply chain.

Conclusions

Each value of the matrix can be compared to the ideal/worst scenario to compare the step to each other and to identify along the product life cycle which step and the relative supplier that needs further measures to improve the product performance. Furthermore, a quantitative value for each indicator related to the product life cycle is calculated and compared with the ideal/worst scenario. The case study on Run On Flat represents the first implementation of the quantitative method of PSIA.

     

Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.     

Assessment of luteal function by ultrasonographic appearance and measurement of corpora lutea in goats

In order to characterize the evolution pattern of the corpora lutea (CL) and to compare luteal function with their ultrasonographic appearance, 37 estrous cycles of Serrana goats (n=22) were studied during breeding season. A daily transrectal ultrasound scanning was performed through two successive estrous cycles. Both solid and fluid-filled CL were observed and measured in both ovaries of each goat. Additionally, each CL was classified as CL(ICHE) (CL with irregular contours and heterogeneous echotexture) or CL(RCGE) (CL with regular contours and granular echotexture). Ovarian cyclic activity and luteal function were evaluated by biweekly plasma progesterone (P4) determination. The CL (n=60) were first visualized on day 2.9+/-1.0 after the day of ovulation (day 0), showing 7.1+/-1.8mm of diameter and reach their maximum size (12.5+/-1.6mm) on day 10.7+/-3.2 (P<0.001). Two days before the following ovulation (day -2), the CL regressed to 8.4+/-1.3mm (P<0.001). The central cavity was found in 78.3% of CL, and had a persistence of over 50% until the last days of estrous cycle. The ratio CL length/cavity length was low during the first-third and high during the remaining two-thirds of estrous cycle. On day 2, the percentage of CL(ICHE) was 33.3%, and began to decrease to 16.7% on day 6, reaching the minimum of 3.3% on day 10 (P<0.001). This proportion increased on day -3 to 48.3% and reached 90% on day -1 (P<0.001). The correlation between CL size and plasma P4 levels was r=0.63 (n=87; P<0.001). A negative correlation between the daily proportion of CL(ICHE) and plasma P4 levels was found (r=-0.95; n=18; P<0.001). These results suggest that the ultrasonographic appearance of CL is a reliable parameter for the assessment of luteal function in goats. Both the characterization of echotexture and size of central cavity could be valuable tools to differentiate between phases of normal estrous cycles.