Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.     

Biological sample collection and processing for molecular epidemiological studies

Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results.Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children's Environmental Health Center, at the University of California, Berkeley.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Association of organophosphate pesticide exposure and paraoxonase with birth outcome in Mexican-American women

BackgroundEpidemiologic studies suggest that maternal organophosphorus (OP) pesticide exposure is associated with poorer fetal growth, but findings are inconsistent. We explored whether paraoxonase (PON1), a key enzyme involved in detoxification of OPs, could be an effect modifier in this association.MethodsThe study population included 470 pregnant women enrolled in the CHAMACOS Study, a longitudinal cohort study of mothers and children living in an agricultural region of California. We analyzed urine samples collected from mothers twice during pregnancy for dialkyl phosphate (DAP) metabolites of OP pesticides. We analyzed maternal and fetal (cord) blood samples for PON1 genotype (PON1₁₉₂ and PON1₋₁₀₈) and enzyme activity (paraoxonase and arylesterase). Infant birth weight, head circumference, and gestational age were obtained from medical records.ResultsInfants'' PON1 genotype and activity were associated with birth outcome, but mothers'' were not. Infants with the susceptible PON1_−108TT genotype had shorter gestational age (β = −0.5 weeks, 95% Confidence Interval (CI): −0.9, 0.0) and smaller head circumference (β = −0.4 cm, 95% CI: −0.7, 0.0) than those with the PON1_−108CC genotype. Infants'' arylesterase and paraoxonase activity were positively associated with gestational age. There was some evidence of effect modification with DAPs: maternal DAP concentrations were associated with shorter gestational age only among infants of the susceptible PON1_−108TT genotype (p-value_interaction = 0.09). However, maternal DAP concentrations were associated with larger birth weight (p-value_interaction = 0.06) and head circumference (p-value_interaction<0.01) in infants with non-susceptible genotypes.ConclusionsInfants whose PON1 genotype and enzyme activity levels suggested that they might be more susceptible to the effects of OP pesticide exposure had decreased fetal growth and length of gestation. PON1 may be another factor contributing to preterm or low birth weight birth.     

Soda consumption and overweight status of 2-year-old mexican-american children in california

Objective: The prevalence of overweight in United States children, 2 to 5 years old, has increased 2‐fold since 1975, with the highest prevalence in Mexican Americans. The objective of this study was to determine the association between current soda consumption and overweight in 2‐year‐old Mexican‐American children. Research Methods and Procedures: The Center for the Health Assessment of Mothers and Children of Salinas study is a longitudinal study of the health of low‐income Latino pregnant women and their children living in the Salinas Valley, CA. Six hundred pregnant women were enrolled (October 1999 to October 2000), and their children were followed until 2 years of age. This cross‐sectional analysis includes the 354 children who completed the 2‐year follow‐up interview. Standing height (centimeters) and weight (grams) were measured at 2 years. Overweight was defined as ≥95th percentile of the sex‐specific BMI for each child's age. Results: Fifty‐five (15.5%) children were overweight. Over half (56%) reported consuming any soda in the last week. After covariate adjustment, compared with no soda consumption, <1 soda/d was not related to overweight (adjusted odds ratio, 0.97; 95% confidence interval, 0.47, 1.99), but ≥1 soda/d was significantly associated with overweight (adjusted odds ratio, 3.39; 95% confidence interval, 1.43, 8.07), and the test for trend was significant (p = 0.02). Discussion: At 2 years of age, the prevalence of overweight among the Center for the Health Assessment of Mothers and Children of Salinas cohort is higher than the national prevalence estimate for Mexican‐American 2‐ to 5‐year‐old children and is significantly associated with current soda consumption. Interventions to reduce consumption of soda in young Mexican‐American children should be considered.     

Predicted warming and browning affect timing and magnitude of plankton phenological events in lakes: a mesocosm study

1. Aquatic ecosystems in Northern Europe are expected to face increases in temperature and water colour (TB) in future. While effects of these factors have been studied separately, it is unknown whether and how a combination of them might affect phenological events and trophic interactions. 2. In a mesocosm study, we combined both factors to create conditions expected to arise during the coming century. We focused on quantifying effects on timing and magnitude of plankton spring phenological events and identifying possible mismatches between resources (phytoplankton) and consumers (zooplankton). 3. We found that the increases in TB had important effects on timing and abundance of different plankton groups. While increased temperature led to an earlier peak in phytoplankton and zooplankton and a change in the relative timing of different zooplankton groups, increased water colour reduced chlorophyll‐a concentrations. 4. Increased TB together benefitted cladocerans and calanoid copepods and led to stronger top‐down control of algae by zooplankton. There was no sign of a mismatch between primary producers and grazers as reported from other studies. 5. Our results point towards an earlier onset of plankton spring growth in shallow lakes in future with a stronger top‐down control of phytoplankton by zooplankton grazers.     

Effects of brown and turbid water on piscivore–prey fish interactions along a visibility gradient

1. Environmental changes such as eutrophication and increasing inputs of humic matter (brownification) may have strong effects on predator–prey interactions in lakes through a reduction in the visual conditions affecting foraging behaviour of visually oriented predators. 2. In this experiment, we studied the effects of visual range (25–200 cm) in combination with optically deteriorating treatments (algae, clay or brown humic water) on predator–prey interactions between pike (Esox lucius) and roach (Rutilus rutilus). We measured effects on reaction distance and strike distance for pike and escape distance for roach, when pike individuals were exposed to free‐swimming roach as well as to roach held in a glass cylinder. 3. We found that reaction distance decreased with decreasing visual range caused by increasing levels of algae, clay or humic matter. The effect of reaction distance was stronger in turbid water (clay, algae) than in the brown water treatment. 4. Strike distance was neither affected by visual range nor by optical treatment, but we found shorter strike distances when pike attacked roach using visual cues only (roach held in a cylinder) compared to when pike could use multiple senses (free‐swimming roach). Escape distance for roach was longer in turbid than in brown water treatments. 5. Changes in environmental drivers, such as eutrophication and brownification, affecting the optical climate should thus have consequences for the strength of predator–prey interactions through changes in piscivore foraging efficiency and prey escape behaviour. This in turn may affect lake ecosystems through higher‐order interactions.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.