Respondent Driven Sampling: Determinants of Recruitment and a Method to Improve Point Estimation

Introduction

Respondent-driven sampling (RDS) is a variant of a link-tracing design intended for generating unbiased estimates of the composition of hidden populations that typically involves giving participants several coupons to recruit their peers into the study. RDS may generate biased estimates if coupons are distributed non-randomly or if potential recruits present for interview non-randomly. We explore if biases detected in an RDS study were due to either of these mechanisms, and propose and apply weights to reduce bias due to non-random presentation for interview.

Methods

Using data from the total population, and the population to whom recruiters offered their coupons, we explored how age and socioeconomic status were associated with being offered a coupon, and, if offered a coupon, with presenting for interview. Population proportions were estimated by weighting by the assumed inverse probabilities of being offered a coupon (as in existing RDS methods), and also of presentation for interview if offered a coupon by age and socioeconomic status group.

Results

Younger men were under-recruited primarily because they were less likely to be offered coupons. The under-recruitment of higher socioeconomic status men was due in part to them being less likely to present for interview. Consistent with these findings, weighting for non-random presentation for interview by age and socioeconomic status group greatly improved the estimate of the proportion of men in the lowest socioeconomic group, reducing the root-mean-squared error of RDS estimates of socioeconomic status by 38%, but had little effect on estimates for age. The weighting also improved estimates for tribe and religion (reducing root-mean-squared-errors by 19–29%), but had little effect for sexual activity or HIV status.

Conclusions

Data collected from recruiters on the characteristics of men to whom they offered coupons may be used to reduce bias in RDS studies. Further evaluation of this new method is required.     

Regulation of Focal Adhesion Kinase Activation,Breast Cancer Cell Motility,and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.     

Mutational analysis of TraM correlates oligomerization and DNA binding with autoregulation and conjugative DNA transfer

F plasmid TraM, an autoregulatory homotetramer, is essential for F plasmid bacterial conjugative transfer, one of the major mechanisms for horizontal gene dissemination. TraM cooperatively binds to three sites (sbmA, -B, and -C) near the origin of transfer in the F plasmid. To examine whether or not tetramerization of TraM is required for autoregulation and F conjugation, we used a two-plasmid system to screen for autoregulation-defective traM mutants generated by random PCR mutagenesis. A total of 72 missense mutations in TraM affecting autoregulation were selected, all of which also resulted in a loss of TraM function during F conjugation. Mutational analysis of TraM defined three regions important for F conjugation, including residues 3-10 (region I), 31-53 (region II), and 80-121 (region III); in addition, residues 3-47 were also important for the immunoreactivity of TraM. Biochemical analysis of mutant proteins indicated that region I defined a DNA binding domain that was not involved in tetramerization, whereas regions II and III were important for both tetramerization and efficient DNA binding. Mutations in region III affected the cooperativity of binding of TraM to sbmA, -B, and -C. Our results suggest that tetramerization is important for specific DNA binding, which, in turn, is essential for traM autoregulation and F conjugation. These findings support the hypothesis that TraM functions as a "signaling" factor that triggers DNA transport during F conjugation.     

Cervical cytology quality assurance in Washington State.

The objectives of this study were to establish a profile of cervical cytology laboratories in Washington State, identify quality assurance problems amenable to correction through education or legislation, and describe differences between large and small cytology laboratories. All 43 Washington laboratories that perform cervical cytology were surveyed by mail during 1989. Completed surveys were returned by 37 (86%) of the laboratories. Nearly half (43%) of the respondents reported processing less than 10,000 Papanicolaou smears annually. Only one-third (35%) of the respondents reported participating in relevant proficiency programs. A proportion of smaller cytology laboratories were compensating their cytotechnologists on the basis of the number of slides read and allowing Papanicolaou smears to be read outside the confines of the laboratory. The results of this study suggest that cytotechnologists in some larger Washington laboratories have been exceeding work load limits recommended by professional associations. Recent legislation includes regulations that address cervical cytology quality assurance. However, continued efforts will need to be made to encourage voluntary adoption of quality control measures not addressed by this legislation.     

A comprehensive model of hyaluronan turnover in the mouse

The metabolism of hyaluronan (HA), especially its catabolism, is still far from being elucidated. Although several studies suggest that HA is degraded locally in tissues and through the lymphatic or circulatory systems, much needs to be learned about the enzymes, receptors and cell types that support this dynamic process. In the current work, the clearance of exogenously administered HA was examined in a C57BL/6 mouse model. Hyaluronidase-sensitive fluorescein-labeled 1.2MDa hyaluronan (flHA) was administered either intravenously (i.v.) or subcutaneously (s.c.) into wild type C57BL/6 mice. Plasma was sampled for pharmacokinetic analysis and tissues were harvested for histological examination of the cell types responsible for uptake using immunofluorescent localization and for size exclusion chromatography analysis. We observed that flHA could be degraded locally in the skin or be taken up by sinusoidal cells in lymph nodes, liver and spleen. I.v. administration of flHA revealed non-linear Michaelis-Menten pharmacokinetics compatible with a saturable, receptor-mediated clearance system (K(m)=11.6μg/ml±46.0%, V(max)=1.69μg/ml/min±59.7%). Through a combination of immunofluorescence microscopy, pharmacokinetic, and chromatographic analyses of labeled substrate in vivo, our results shed additional light on the mechanisms by which HA is catabolized in mammals, and serve as a basis for future studies.     

The neonate nutrition hypothesis: early feeding affects the body stoichiometry of Daphnia offspring

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Immunohistochemical Evaluation of Global DNA Methylation: Comparison with in Vitro Radiolabeled Methyl Incorporation Assay

The in vitro radiolabeled methyl incorporation assay, a commonly used technique to evaluate global methylation of DNA, has some disadvantages and limitations. The purpose of the present study was to compare the results of global DNA methylation evaluated by radiolabeled methyl incorporation (CPM/μg of DNA) with immunohistochemical staining of the same tissue sections with a monoclonal antibody developed against 5-methylcytosine (5-mc). We used archival specimens of squamous cell cancer (SCC) of the human lung with a matched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjects without lung cancer (noncancer specimens) to make this comparison. The immunostaining for 5-mc was reported as a percentage of cells positive for staining as well as a weighted average of the intensity score. The results suggested that both radiolabeled methyl incorporation assay and immunostaining for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissues compared to matched uninvolved tissues. An advantage of immunostaining, however, is its ability to demonstrate hypomethylation of SCC compared to adjacent bronchial mucosa on the same archival specimen, obviating the need to use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically significant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolabeled methyl incorporation assay and may be best suited for evaluation of global DNA methylation when the methylation status of cancer cannot be normalized by methyl incorporation of normal tissues or when the number of samples available for evaluation is small.     

Site-specificity of histone H1 methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. When the enzymatically [methyl-3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (H1b, H1c, H1d and H1e). Both enzymes methylated H1c and H1b to approximately the same extent; H1d and H1e were methylated preferentially by enzyme V-B and V-A, respectively. 3. Histone H1c, [methyl-3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. Arg C protease-digestion of [methyl-3H]labeled H1c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. The histone H1c methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.     

Trees on networks: resolving statistical patterns of phylogenetic similarities among interacting proteins

Background  

Phylogenies capture the evolutionary ancestry linking extant species. Correlations and similarities among a set of species are mediated by and need to be understood in terms of the phylogenic tree. In a similar way it has been argued that biological networks also induce correlations among sets of interacting genes or their protein products.     

Predator-induced diet vertical migration in a planktonic copepod

Predator evasion is the most commonly hypothesized reason fordiel vertical migrations undertaken by a wide variety of planktonicorganisms in lakes and seas, yet direct evidence remains elusive.We tested the predation hypothesis by exposing enclosed populationsof a marine copepod Acartia hudsonica to caged or free-rangingindividuals of their natural predator, the planktivorous fishGasterosteus aculeatus. After little more than a week, adultcopepods changed their vertical distribution and migration behaviordepending on the presence or absence of predation. Only free-rangingfish induced vertical migration in the copepod population. Cagedfish had no effect, indicating that vertical migration was nota simple chemically mediated response of copepods to the predator.Rather, copepods seemed to react to the presence of predatorsby other means, perhaps visual or mechanical stimuli, and toexhibit a downward escape response which, because encounterswith visually orienting fish occur chiefly in the daytime, effectivelylimited the copepods' occurrence in the upper water column tothe night-time hours. Alternatively, because fish imposed heavymortality on copepods, it is possible that selective predationaltered the proportions of individuals with fixed, geneticallydetermined migration behaviors. We suggest experiments to distinguishthese alternatives.