Development of a new peptide-bead coupling method for an all peptide-based Luminex multiplexing assay for detection of Plasmodium falciparum antibody responses.

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas.

The repeatability of vegetation classification and mapping.

The mapping of habitats as defined by plant communities is a common component of the planning and monitoring of conservation management. However, there are major concerns about the subjectivity and risk of observer bias in most commonly used plant community mapping protocols. This study provides the first test of the consistency of habitat maps based on the mapping units defined by the National Vegetation Classification (NVC), the most widely used classification of plant communities used for habitat mapping on conservation sites in the UK. Seven surveyors mapped the same upland site within five weeks in summer 2008 and the spatial correspondence of the resulting maps was assessed. The NVC is a hierarchical classification and pair-wise spatial agreement between maps decreased with lower levels of sub-classification. The average area of agreement between maps was 77.6% at the habitat level, 34.2% at the community level and 18.5% at the sub-community level. Spatial disparity in the location of mapped boundaries between vegetation types only made a small contribution to overall differences; the majority of variation between maps was due to discrepancies in classification, with vegetation types containing similar species composition most often confused. Factors relating to surveyor effort (cost, time taken and length of route) were not able to explain the substantial differences between maps. However, the methods used to assign areas to vegetation type did seem to have an effect, with surveyors who relied primarily on their own experience having the highest levels of mean agreement with other maps. The study raises serious concerns with current practice of using the NVC for site description and monitoring/surveillance. Since this is just a single case study, we recommend that further work is carried out with the aim of determining the degree and source of variation between surveyors and how consistency can be increased.

Sirolimus is associated with impaired hematopoiesis in heart transplant patients? A retrospective analysis.

BACKGROUND: Proliferation signal inhibitors may adversely impact bone marrow function. We sought to describe the impact of sirolimus on hemoglobin and erythropoiesis in heart transplant recipients. METHODS: We have conducted a single-center, retrospective analysis of all heart transplant patients treated with sirolimus. We measured serum hemoglobin (Hb) at baseline and at 3 months to determine the prevalence of anemia and change in Hb after sirolimus initiation. We also characterized hematologic profile of patients to gain insights into the effects of sirolimus on erythropoiesis. RESULTS: There were 84 patients included in the study. The prevalence of anemia increased from 71% to 75% after sirolimus initiation. Anemic patients were more likely to be male (P = .026) and have worse renal function (glomerular filtration rate 49 ± 27 vs 70 ± 42 mL/min; P = .012). A ≥20 g/L drop in Hb was observed in 25% of the overall cohort. Patients investigated for anemia (n = 67) had a low Hb (111 ± 24 g/L), normal mean corpuscular volume (87 ± 47 FL), and low serum iron levels (10 ± 5 µmol/L) and transferrin saturation (0.22 ± 0.12). Serum ferritin was variable (263 ± 370 µg/L). Bone marrow evaluation in 19 patients revealed adequate marrow iron stores in all cases. CONCLUSION: Anemia is prevalent in heart transplant patients treated with sirolimus and increases over time. Patients have a characteristic hematologic profile suggestive of anemia of chronic disease and functional iron deficiency.

Are we failing vulnerable workers? The case of black women in poultry processing in rural North Carolina.

In 1989, North Carolina Occupational Safety and Health Administration (OSHA) inspectors cited two poultry processing plants in northeastern North Carolina for serious repetitive motion problems. In 1990, investigators from the National Institute for Occupational Safety and Health confirmed significant upper extremity musculoskeletal symptoms and disorders among workers. We now report on analyses of baseline data collected from a cohort of women employed in one of these plants. The plant, which is the largest employer of women in the area, is located in a sparsely populated area with a black majority where nearly one-third of the population lives below the poverty level. Conditions we report suggest failure of existing health and safety systems, both regulatory and consultative, to prevent morbidity among vulnerable women in this industry, as well as social and economic conditions that influence availability of work and use of benefits to which they are entitled.

Work related injuries in small scale commercial fishing.

OBJECTIVE: To describe the epidemiology of work related injury in a group of small scale, independent commercial fishers. DESIGN: Cross sectional survey (baseline instrument of a prospective cohort study). SETTING AND SUBJECTS: Commercial fishers in eastern North Carolina. RESULTS: A cohort of 219 commercial fishers was established and 215 subjects completed an injury questionnaire. The main types of fishing conducted by the cohort were finfishing (159/215) and crabbing (154/215). Of the 215 fishers, 83 reported that they had suffered an injury event in the previous 12 months, a retrospective recall incidence proportion of 38.6 per 100 workers (95% confidence interval 32.1 to 45.1). The 83 injury events resulted in 94 injuries; 47% were penetrating wounds and 24% were strains/sprains. Half of injuries were to the hand/wrist/digits and 13% were to the back. Of the penetrating wounds, 87% were to the hand/wrist/digits, 32% became infected, and 80% were caused by contact with finfish, shellfish, or other marine animal. Of the strains/sprains, 48% were to the back and 26% were to the shoulder. Seventy percent of strains/sprains were caused by moving heavy objects, mainly either while hauling in nets, pots, or lines or loading/unloading the boat. CONCLUSION: In this group of small scale, independent fishers, the most common reported injuries were penetrating wounds to the hand/wrist/digits from marine animals and strains/sprains to the back while moving heavy objects.

When is sex safe? Insiders' views on sexually transmitted disease prevention and treatment.

STD prevention programs promote practices and messages that are presumed to fit with most conditions of U.S. communities. Yet, the social and cultural contexts for low-income ethnic communities may frame STD prevention differently, so that people calculate their risk and take actions based on what they have learned through their own observations and life experiences. To understand how people at high risk for STDs make decisions and take actions to protect themselves from these diseases, the authors conducted 38 ethnographic interviews with individuals living in arural community in the South. Practices they reported include selecting "safe" partners on the basis of appearance, familiarity, or church attendance; washing before and after sex to prevent infection; self-treatment with antibiotics obtained without a prescription; and visiting the clinic frequently for checkups for asymptomatic infections. The authors compare and contrast their worldview with the public health concepts of primary and secondary STD prevention.

The acoustic calls of blue whales off California with gender data.

The acoustic calls of blue whales off California are described with visual observations of behavior and with acoustic tracking. Acoustic call data with corresponding position tracks are analyzed for five calling blue whales during one 100-min time period. Three of the five animals produced type A-B calls while two produced another call type which we refer to as type D. One of the animals producing the A-B call type was identified as male. Pauses in call production corresponded to visually observed breathing intervals. There was no apparent coordination between the calling whales. The average call source level was calculated to be 186 dB re: 1 muPa at 1 m over the 10-110-Hz band for the type B calls. On two separate days, female blue whales were observed to be silent during respective monitoring periods of 20 min and 1 h.

Evaluating fitness to drive after cerebral injury: basic issues and recommendations for medical and legal communities.

Specialists in rehabilitation are typically called upon to evaluate and render an opinion about whether or not a person can be entrusted to resume driving. And, because driving is an individual privilege to be balanced against the public's right to safety and protection from the dangers of a driver whose residual deficits may impede ability to drive safely, these specialists have developed a number of methods to assess fitness to drive. Unfortunately, many evaluators remain unfamiliar with research used as basis for evaluations or lack understanding of the strengths and weaknesses of tests in use. Therefore, there may be unquestioning trust in tests and methods that leads to errors of significant consequence in decisions about fitness to drive as well as unawareness of expanding risks of litigation that can emanate from inappropriate recommendations. This article intends to draw attention to issues, considerations, and problems underlying the conduct of driver evaluations, including focus on ways in which the legal and medical communities approach question of fitness, legal and medical definitions and terminology, responsibility for assessment as well as tests and methods used in evaluations. Conclusions are drawn from discussion of these matters and recommendations are outlined for addressing identified problems at the interface between medical and legal communities.

How to keep scuba diving safe.

As scuba diving grows in popularity, divers must know the safety factors involved. For example, they need to know their abilities and how to dive within those limits, how to react to emergency situations, and how to prevent and treat common medical conditions. Below are clear answers to common questions.

Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: a study of tyrosine-to-phenylalanine mutants.

Three tyrosine-to-phenylalanine mutants of ribonuclease A (Y25F, Y92F, and Y97F) are investigated for their enzymatic activities, molecular stabilities, and unfolding/refolding kinetics. These mutants exhibit 80, 90, and 80%, respectively, of the catalytic activity of the wild-type enzyme. Thermal, Gdn.HCl, and pH transition measurements indicate that Y25F and Y97F are less stable than the wild-type protein, whereas the bulk of the thermodynamic and kinetic evidence indicates that Y92F is as stable as the wild-type protein. Differences in molar extinction coefficients indicate that tyrosines 25, 92, and 97 contribute 38, 13, and 39%, respectively, to the absorption difference between the folded and unfolded states, in general agreement with previous studies but possibly indicating the contribution of a fourth tyrosine residue to account for the remaining 10%. Stopped-flow single- and double-jump kinetic experiments were carried out on the wild-type and three mutant proteins. At least one tyrosine residue besides tyrosine 92 contributes to the slow fluorescence-unfolding phase; the likely candidate for this residue is tyrosine 115 which monitors the cis-trans isomerization of the X-Pro114 peptide bond. Tyrosines 25 and 97 are involved in interactions that retard conformational unfolding and accelerate conformational refolding as well as the cis-trans proline isomerization of the slow-refolding phases, presumably by stabilizing the major beta-hairpin structure of RNase A. These interactions may contribute to the strong pH dependence of the folding and unfolding of ribonuclease A. In contrast, tyrosine 92 does not affect the folding and unfolding rates significantly. An improved "box" model of proline isomerization under unfolding conditions was derived from exhaustive fitting of all possible box models. The kinetic data support the identification of Pro93 as the proline whose isomerization distinguishes the slow-refolding species (USII and USI) from the other, faster-refolding species (Uvf, Uf, and Um), implying that Pro93 isomerizes in the slow-refolding reactions USI --> N and IN --> N. Similarly, Pro114 seems to distinguish between the very fast-refolding species Uvf and the fast-refolding species Uf. Lastly, Pro117 seems to distinguish the major slow-refolding species USII from the minor slow-refolding species USI and the medium-refolding species Um from the fast- and very fast-refolding species.

Male student-athletes and violence against women: a survey of campus judicial affairs offices.

PIP: This study examines the institutional dimensions of violence against women through an analysis of officially reported incidents of violence against women. The data included records from 10 judicial affairs offices over a 3-year period from 1991-93. The findings reveal that male college student-athletes, compared to the rest of the male student population, are responsible for more than their share of the reported battering and sexual assault complaints to judicial affairs offices on the campuses of 10 Division I institutions. However, it needs to be emphasized that it is uncertain whether the association between athletic affiliation and violence against women is casual or the result of behavior only indirectly related to sport. These findings relate to other research because they are based on official reports and they strengthen previous works on athletes and violence against women that center on single institutions.^ieng

A treatment and withdrawal trial of besipirdine in Alzheimer disease.

Besipirdine hydrochloride (HP 749) is an indole-substituted analog of 4-aminopyridine. Besipirdine enhances both cholinergic and adrenergic neurotransmission in the central nervous system. The present study examined the efficacy and tolerability of two doses of besipirdine (5 and 20 mg b.i.d.) in 275 patients with Alzheimer disease during 3 months of treatment and for 3 months after withdrawal of treatment. Assessment after withdrawal of treatment was used in an effort to distinguish persistent efficacy attributable to a neuroprotective mechanism from reversible symptomatic efficacy. Besipirdine was generally well tolerated. The level of performance on the cognitive subscale of the Alzheimer Disease Assessment Scale (ADAS-Cog) was sustained during 3 months of treatment with besipirdine, whereas some deterioration in the performance of patients treated with placebo was observed over the same period. The small difference between active and placebo treatment groups approached, but did not reach statistical significance in the primary intent-to-treat analysis (p = 0.067); analysis of patients who completed all assessments was supportive (p = 0.031). Global ratings using the Clinician Interview-Based Impression of Change did not detect a besipirdine treatment benefit, possibly because of an adverse effect on mood and behavior in some patients. A high ratio of adrenergic to cholinergic potency may have resulted in the adverse effects of besipirdine and hence its failure to support the hypothesis that multiple neurotransmitter treatment may be more efficacious than monotherapy. The efficacy apparent on the ADAS-Cog after 3 months of treatment did not persist 3 months after withdrawal of treatment, suggesting that the benefit was symptomatic. This study provides a practical example of the use of treatment withdrawal assessment to distinguish neuroprotective from symptomatic efficacy.

Blue and fin whales observed on a seafloor array in the northeast pacific.

Calling blue and fin whales have been tracked using relative travel times and amplitudes from both direct and multipath arrivals to a seafloor array of seismometers. Calls of three fin whales swimming in the same general direction, but several kilometers apart, are believed to represent communication between the whales because of signature differences in call character, an alternating call pattern, and coordination of call and respiration times. Whale call tracks, call patterns, call character, and swimming speeds were examined during periods with and without the presence of noise. Noise sources included airguns, when the whales were subject to sound levels of up to 143 dB P-P (peak-to-peak) re: 1 microPa over the 10 to 60-Hz band, and transits of merchant ships, when the whales received continuous levels up to 106 dB rms re: 1 microPa over the 10 to 60-Hz band (115 dB P-P). Whale responses associated with these noises remain arguable.

Lymphocytic hypophysitis: a rare cause of hyperprolactinaemia.

Lymphocytic hypophysitis is one of the rare causes of hypopituitarism and intrasellar mass observed almost exclusively in women. A temporal relationship to pregnancy has been identified in many but not all of the 34 reported cases. Intra-operative differentiation from non-secretory macroadenoma is important, as resection of the anterior lobe is unnecessary in these individuals and pituitary function may spontaneously return to normal. The authors present the case of a woman with symptomatic hyperprolactinaemia 10 years after her last pregnancy.

Impact of a temporary food shortage on children and their mothers.

Kenya experienced a severe drought and temporary food shortage during a study on mild malnutrition. Effects of the temporary food shortage on energy intake, weight, and behaviors were evaluated in schoolchildren and in toddlers and their mothers. Schoolchildren were seriously affected, showing significant declines in their energy intake, age-corrected weight, activity on the playground, and classroom attention. Toddlers appear to have been somewhat protected since their energy intake, weight, and play and language behaviors were stable. Maternal caregiving of toddlers declined for the group as a whole, but individually those mothers who maintained family food levels delegated responsibility for toddlers to other caregivers. While the food shortage affected poorer families more than those of higher SES, declines in the behaviors of schoolchildren occurred regardless of SES and previous level of nutrition, suggesting that food shortages can have behavioral consequences for schoolchildren in all communities.

Differentiation of species and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD).

Polymerase chain reaction (PCR)-based technology, involving random amplification of polymorphic DNA (RAPD), was used to assess the genomic variability between 24 isolates of deuteromycetous fungi (Metarhizium anisopliae, Metarhizium flavoviride, unidentified strains of Metarhizium and Beauveria bassiana) which were found to infect grasshoppers or locusts. M. flavoviride showed little intraspecific variability in PCR-amplified fragments when compared to M. anisopliae. The high level of variability in PCR-amplified fragments contained within M. anisopliae was similar to the total variability between B. bassiana, M. anisopliae and M. flavoviride, and suggests that M. anisopliae may include a number of cryptic species. Four polymorphic RAPD fragments were used to probe the genomic DNA of the various species and strains. On the basis of these probes the fungi can be grouped into M. flavoviride, M. anisopliae, or B. bassiana. According to PCR-amplified fragments, previously-unidentified Metarhizium strains were characterized as M. flavoviride. There was little evidence that these fungi, all isolated from, or virulent towards, grasshoppers or locusts, showed host-selection in PCR-amplified fragments. Nor was geographical origin a criterion for commonality based on PCR-amplified fragments. PCR-fragment-pattern polymorphisms and the construction of probes from one or more of these fragments may provide a useful and rapid tool for identifying species and strains of entomopathogenic fungi.

Medical illness in school-age Kenyans in relation to nutrition, cognition, and playground behaviors.

Because medical illness is associated with malnutrition, it might contribute to the poor outcomes seen in malnourished children. This study explored relations between frequency of mild illness and development in 133 mild-to-moderately malnourished school-age Kenyan children. Morbidity information was collected for one year. Concurrent assessments of cognitive status and playground behaviors were obtained. Nutritional and environmental variables were also assessed. Girls with more reported days of mild illness performed less well on developmental measures than did their healthier female peers. They were less cognitively advanced, and, on the playground, girls with more mild illness were less active, happy, and social. Furthermore, relations between frequency of mild illness and playground behaviors (but not cognitive score) in the girls remained statistically significant when other variables, such as socioeconomic status, parental literacy, food intake, and anthropometry, were considered. For the boys, frequency of mild illness was not related to cognition or play behaviors.

Relationships between morbidity and development in mildly to moderately malnourished Kenyan toddlers.

This study explored the extent to which morbidity in 110 mildly to moderately malnourished Kenyan toddlers was associated with developmental outcomes. Morbidity information was collected from the 18th to 30th months. Concurrent assessments of vocalization, play, and performance on the Bayley scales were obtained. At 5 years, a follow-up battery of cognitive tests was administered. Female toddlers who suffered more illness generally performed less well on developmental measures than their healthier female peers. These children vocalized and played less and performed less well on the Bayley Mental scales at 30 months and on the cognitive battery at 5 years. For the boys, development was largely independent of morbidity. Morbidity was related to patterns of care giving for both boys and girls, but it was not associated with socioeconomic status or food intake. However, girls who were ill more often were shorter and lighter. Relations between morbidity and development in the girls remained statistically significant when other variables, which were also related to development (such as care giving, socioeconomic status, parental IQ and literacy, food intake, and anthropometry) were considered. This suggests that morbidity, in these female toddlers, had an effect on development above and beyond other variables typically associated with malnutrition.

Unreliability of standard quantitative criteria in diagnostic peritoneal lavage performed for suspected penetrating abdominal stab wounds.

Thirty-five patients with abdominal stab wounds in whom clinical examination was equivocal on 2 separate occasions underwent diagnostic peritoneal lavage (DPL) prior to laparotomy. The red and white blood cell counts (cells/mm3) of the lavage effluent were compared with the operative findings. There were 26 positive and 9 unnecessary laparotomies, the latter consisting of 4 negative and 5 non-therapeutic operations. Use of the standard quantitative criteria for red cells in DPL failed to identify significant injury in eight patients (31%), while the standard white cell count missed six injuries (23%). Their combined use resulted in three missed injuries (12%). Two false-positive results occurred using the red cell count alone and four using the white cell count alone, producing a combined false-positive result in four patients (11%). Reducing the cell threshold level to exclude missed injuries would increase dramatically the rate of unnecessary laparotomies. Although the standard quantitative criteria for DPL are superior to clinical assessment in patients with equivocal findings, their use in penetrating trauma does not achieve the same diagnostic accuracy as in blunt abdominal trauma.