To What did They Consent? Understanding Consent Among Low Literacy Participants in a Microbicide Feasibility Study in Mazabuka,Zambia

We conducted a study to review the consenting process in a vaginal Microbicide feasibility study conducted in Mazabuka, Zambia. Participants were drawn from those participating in the microbicide study. A questionnaire and focus group discussion were used to collect information on participants understanding of study aims, risks and benefits. Altogether, 200 participants took part in this study. The results of the study showed that while all participants signed or endorsed their thumbprints to the consent forms, full informed consent was not attained from most of the participants since 77% (n = 154) of the participants had numerous questions about the study and 34% (n = 68) did not know who to get in touch with concerning the study. Study objectives were not fully understood by over 61% of the participants. Sixty four percent of the participants were not sure of the risks of taking part in the microbicide study. A significant number thought the study was all about determining their HIV status. Some participants were concerned that their partners were not on the trial as they were convinced that being on the study meant that that they had a lifetime protection from HIV infection. The process of obtaining consent was inadequate as various phases of the study were not fully understood. We recommend the need for researchers to reinforce the consenting process in all studies and more so when studies are conducted in low literacy populations.     

Persistent Posttetanic Depression at Cerebellar Parallel Fiber to Purkinje Cell Synapses

Plasticity at the cerebellar parallel fiber to Purkinje cell synapse may underlie information processing and motor learning. In vivo, parallel fibers appear to fire in short high frequency bursts likely to activate sparsely distributed synapses over the Purkinje cell dendritic tree. Here, we report that short parallel fiber tetanic stimulation evokes a ∼7–15% depression which develops over 2 min and lasts for at least 20 min. In contrast to the concomitantly evoked short-term endocannabinoid-mediated depression, this persistent posttetanic depression (PTD) does not exhibit a dependency on the spatial pattern of synapse activation and is not caused by any detectable change in presynaptic calcium signaling. This persistent PTD is however associated with increased paired-pulse facilitation and coefficient of variation of synaptic responses, suggesting that its expression is presynaptic. The chelation of postsynaptic calcium prevents its induction, suggesting that post- to presynaptic (retrograde) signaling is required. We rule out endocannabinoid signaling since the inhibition of type 1 cannabinoid receptors, monoacylglycerol lipase or vanilloid receptor 1, or incubation with anandamide had no detectable effect. The persistent PTD is maximal in pre-adolescent mice, abolished by adrenergic and dopaminergic receptors block, but unaffected by adrenergic and dopaminergic agonists. Our data unveils a novel form of plasticity at parallel fiber synapses: a persistent PTD induced by physiologically relevant input patterns, age-dependent, and strongly modulated by the monoaminergic system. We further provide evidence supporting that the plasticity mechanism involves retrograde signaling and presynaptic diacylglycerol.     

A TILLING Platform for Functional Genomics in Brachypodium distachyon

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called “BRACHYTIL”, for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.     

Polygenic inheritance of resistance to <Emphasis Type="Italic">Cacopsylla pyri</Emphasis> in a <Emphasis Type="Italic">Pyrus communis</Emphasis> × <Emphasis Type="Italic">P. ussuriensis</Emphasis> progeny is explained by three QTLs involving an epistatic interaction

Pear psylla (Cacopsylla pyri) causes severe damage on European pear cultivars, resulting in high yield losses. Its control has become difficult since it developed resistance to a wide range of pesticides, while the number of authorized molecules for pest control has decreased. Identifying pear psylla resistance factors should help breeding new resistant pear cultivars. We analyzed the quantitative resistance to psylla inherited from the genotype NY 10355 derived from Pyrus ussuriensis. Quantitative trait locus (QTL) analysis was carried out after counting the number of nymphs and estimating the nymphal development rate using a free-choice test performed on a large segregating progeny. We mapped two new loci for pear psylla resistance on linkage groups LG01 and LG04 of NY 10355 and confirmed the QTL previously detected on LG17. A strong epistatic interaction between the two QTLs detected on LG01 and LG17 appeared to be a major factor controlling the psylla infestation in the genotype NY 10355.     

Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.     

Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human delta opioid receptor

Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes that include protein folding, post-translational modifications, and transport through distinct cellular compartments. Relatively little is known about the nature and kinetics of specific steps involved in these processes. Here, the human delta opioid receptor expressed in human embryonic kidney 293S cells is used as a model to delineate these steps and to establish the kinetics of receptor synthesis, glycosylation, and transport. We found that the receptor is synthesized as a core-glycosylated M(r) 45,000 precursor that is converted to the fully mature M(r) 55,000 receptor with a half-time of about 120 min. In addition to trimming and processing of two N-linked oligosaccharides, maturation involves addition of O-glycans containing N-acetylgalactosamine, galactose, and sialic acid. In contrast to N-glycosylation, which is initiated co-translationally and is completed when the protein reaches the trans-Golgi network, O-glycosylation was found to occur only after the receptor exits from the endoplasmic reticulum (ER) and was terminated as early as the trans-Golgi cisternae. Once the carbohydrates are fully processed and the receptor reaches the trans-Golgi network, it is transported to the cell surface in about 10 min. The exit from the ER was found to be the limiting step in overall processing of the receptor. This indicates that early events in the folding of the receptor are probably rate-limiting and that receptor folding intermediates are retained in the ER until they can adopt the correct conformation. The overall low efficiency of receptor maturation, less than 50% of the precursor being processed to the fully glycosylated protein, further suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER. This indicates that folding and ER export are key events in control of receptor cell surface expression. Whether or not the low efficiency of the ER export is a general feature among G protein-coupled receptors remains to be investigated.