Risk and Decision-Making: A Workshop for Environmental Professionals

In the early 1990s, U.S. Environmental Protection Agency Region 9 developed a training workshop for environmental professionals. It was successfully taught throughout Region 9 in collaboration with the California Department of Toxic Substances Control. We have updated the workshop's manual to incorporate current practices including: vapor intrusion into indoor air, benchmark dose, cancer guidelines, inhalation guidance, ecological and screening risk assessments, conceptual site models, and data quality objectives. We maintained the popular workshop format, with participants evaluating information and drawing conclusions in an interactive hands-on approach. We kept the case study approach to simulate realistic environmental issues. After a case study introduction, participants plan a sampling strategy. Principles of toxicology are introduced, and participants develop toxicity criteria using hypothetical animal study results. Participants then identify exposure pathways, and calculate exposure and risk and hazard estimates. Finally, participants develop remedial alternatives and practice risk communication through role playing exercises. The workshop has been an effective tool for training new employees and providing continuing education for experienced employees from consulting, military, and regulatory agencies. The format provides a dynamic learning environment, fostering exchanges among professionals with a wide range of skills and backgrounds (project managers, toxicologists, geologists, engineers, public participation experts).     

Routine procedures for isolation and identification of enterococci and fecal streptococci.

Over the past 6 years, a revised classification of the streptococci and enterococci, based primarily on molecular techniques such as 16S rRNA sequencing and DNA-DNA hybridization, emerged. However, little attention was placed on routine physiological tests that could be used in food and clinical laboratories to differentiate between species of a new genus, Enterococcus, and fecal Streptococcus spp. The purpose of this study was to devise a convenient and reliable system to identify enterococci and fecal streptococci by using conventional procedures. Fifty-nine strains of 13 Enterococcus spp., including the type strains and many strains used by previous investigators, were characterized by using conventional tube tests, the API Rapid Strep system, and MicroScan Pos ID panels. Results were compared with each other and with previously published results. A comparison of conventional tube tests versus published tube test results yielded 17 discrepancies. Although not all tests were done with each of the three systems, 28 discrepancies between results obtained with the API system and those obtained with conventional tube tests were found. There were 24 discrepancies between results obtained with the MicroScan Pos ID panel and those obtained with conventional tube tests. There were 12 discrepancies between the results with the API Rapid Strep system and those with the MicroScan Pos ID panels. We devised flow charts of key tests that might be used to identify cultures without resorting to nucleic acid analysis and other labor- and equipment-intensive analyses.     

Applying instructional design theories to bioinformatics education in microarray analysis and primer design workshops

The need to support bioinformatics training has been widely recognized by scientists, industry, and government institutions. However, the discussion of instructional methods for teaching bioinformatics is only beginning. Here we report on a systematic attempt to design two bioinformatics workshops for graduate biology students on the basis of Gagne's Conditions of Learning instructional design theory. This theory, although first published in the early 1970s, is still fundamental in instructional design and instructional technology. First, top-level as well as prerequisite learning objectives for a microarray analysis workshop and a primer design workshop were defined. Then a hierarchy of objectives for each workshop was created. Hands-on tutorials were designed to meet these objectives. Finally, events of learning proposed by Gagne's theory were incorporated into the hands-on tutorials. The resultant manuals were tested on a small number of trainees, revised, and applied in 1-day bioinformatics workshops. Based on this experience and on observations made during the workshops, we conclude that Gagne's Conditions of Learning instructional design theory provides a useful framework for developing bioinformatics training, but may not be optimal as a method for teaching it.     

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.     

Microbial quality of tropical inland waters and effects of rainfall events

Novel markers of fecal pollution in tropical waters are needed since conventional methods recommended for other geographical regions may not apply. To address this, the prevalence of thermotolerant coliforms, enterococci, coliphages, and enterophages was determined by culture methods across a watershed. Additionally, human-, chicken-, and cattle-specific PCR assays were used to identify potential fecal pollution sources in this watershed. An enterococcus quantitative PCR (qPCR) assay was tested and correlated with culture methods at three sites since water quality guidelines could incorporate this technique as a rapid detection method. Various rainfall events reported before sample collection at three sites were considered in the data analyses. Thermotolerant coliforms, enterococci, coliphages, and enterophages were detected across the watershed. Human-specific Bacteroides bacteria, unlike the cattle- and chicken-specific bacteria, were detected mostly at sites with the corresponding fecal impact. Enterococci were detected by qPCR as well, but positive correlations with the culture method were noted at two sites, suggesting that either technique could be used. However, no positive correlations were noted for an inland lake tested, suggesting that qPCR may not be suitable for all water bodies. Concentrations of thermotolerant coliforms and bacteriophages were consistently lower after rainfall events, pointing to a possible dilution effect. Rainfall positively correlated with enterococci detected by culturing and qPCR, but this was not the case for the inland lake. The toolbox of methods and correlations presented here could be potentially applied to assess the microbial quality of various water types.     

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.     

Monitoring and evaluation of the activities of trainees in the ‘training of trainers’ workshop at Ibadan,south-west Nigeria

Background Like most low- and middle-income countries, Nigeria has a huge treatment gap for mental disorders. The World Health Organization has proposed the integration of mental health care into primary health service delivery as one of the ways to bridge this treatment gap. Studies have shown an immediate positive impact of mental health training for primary care workers. We evaluated the impact of training on the tutors of primary care workers approximately 12 months after the training.Method An intensive five-day training workshop for college teachers of mental health in community health officer (CHO) training institutions in south-west Nigeria was conducted in January 2009. Four of the 24 participants were randomly selected for evaluation of the impact of training on their activities approximately 12 months after the workshop. Qualitative methods were used, namely in-depth interviews, direct observation of classroom teaching by the participants and focus group discussion with their students.Results The participants interviewed reported a positive impact of the ‘training of trainers’ (TOT) workshop on their mental health course teaching. Direct observation of four participants revealed that three of them exhibited a high fidelity with the TOT course material and imbibed the teaching techniques advocated. The tutors'' students also reported an improvement in the quality of their mental health classes.Conclusion The training had an overall positive impact on the activities of the trainees approximately one year after the workshop.     

Comparison of the MSRV method with various rapid and conventional Salmonella detection methods for chocolate, confectionery and biscuit ingredients

The efficacy of MSRV, ELISA, Bactometer, IOCCC and in-house conventional methods for the detection of Salmonella was determined using both naturally contaminated and inoculated food samples. Forty-three food samples were examined at each of three laboratories. No statistically significant difference in the sensitivity of the methods was observed, suggesting the MSRV method to be a suitable rapid, sensitive method for the detection of Salmonella in the range of food samples tested.     

Experiences and lessons learned from two virtual,hands-on microbiome bioinformatics workshops

In October of 2020, in response to the Coronavirus Disease 2019 (COVID-19) pandemic, our team hosted our first fully online workshop teaching the QIIME 2 microbiome bioinformatics platform. We had 75 enrolled participants who joined from at least 25 different countries on 6 continents, and we had 22 instructors on 4 continents. In the 5-day workshop, participants worked hands-on with a cloud-based shared compute cluster that we deployed for this course. The event was well received, and participants provided feedback and suggestions in a postworkshop questionnaire. In January of 2021, we followed this workshop with a second fully online workshop, incorporating lessons from the first. Here, we present details on the technology and protocols that we used to run these workshops, focusing on the first workshop and then introducing changes made for the second workshop. We discuss what worked well, what didn’t work well, and what we plan to do differently in future workshops.     

Influence of handling and conditioning protocol on learning and memory of Microplitis croceipes

Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) learns odors in association with both hosts and food. The food‐associated ‘seeking’ behavior of M. croceipes was investigated under various training protocols utilizing the conditioning odor, 3‐octanone. We investigated the effects of odor training, or its lack, training duration, training frequency, time elapsed after training, wasp hunger state, and training reinforcement, on the food‐seeking responses of M. croceipes females. We found that odor‐trained females show strong food seeking responses, whereas non‐odor‐trained females do not respond to the odor, and that a single 10 s association with the odor whilst feeding on sugar water subsequently conditioned the wasps to exhibiting significant responses to it. Increases in training time to more than 10 s did not improve their responses. Repetition of the food–odor associations increased a wasp's recall, as well as its response over time, compared to a single exposure. Repeated exposure to the learned odor in the absence of a food reward decreased the responses of less hungry individuals. However, the level of response increased significantly following a single reinforcement with the food–odor association. Understanding the factors that influence learning in parasitoids can enhance our ability to predict their foraging behavior, and opens up avenues for the development of effective biological detectors.     

Comparative analysis of comparative genomic hybridization microarray technologies: report of a workshop sponsored by the Wellcome Trust

BACKGROUND: Array-comparative genomic hybridization (CGH), although providing much higher resolution compared with conventional CGH, has not yet become a widely applied method for the analysis of genomic gains and losses. METHODS: In January 2002, the Wellcome Trust sponsored a workshop where many of the laboratories developing this technology met to compare different methodologies for array-CGH. Fourteen groups participated, comprising 11 from Europe and 3 from the United States. To facilitate objective analysis, each laboratory constructed arrays using the same anonymous clones and performed a series of test hybridizations using identical genomic DNAs. RESULTS: A figure of merit (FM) was developed to summarize entire collections of data from each laboratory in a single measurement. The FMs consistently showed that a few groups produced quantitative array hybridization data of high quality, whereas a majority achieved a lower standard. CONCLUSIONS: The conclusions of the workshop were that polymerase chain reaction-based methods for the amplification of large insert clones for arraying were effective for array-CGH. It was also concluded that hybridizations performed under coverslips or in automated hybridization apparatus were less effective than hybridizations performed in simple wells with gentle rocking. A common experience by the participants was the batch-to-batch variability of commercial Cot1 preparations in their ability to suppress hybridization to repeat sequences. (Supplementary material for this article can be found in the online issue, which is available at http://www.interscience.wiley.com/jpages/0196-4763/suppmat/49_2/v49.43.html or at http://www.sanger.ac.uk/HGP/Cytogenetics/Publications/Cytometry Sept 2002/Supplemental.pdf.)     

Preface

Recent studies on food quality for zooplankton have developed two new hypotheses, the importance of the dietary carbon to phosphorus (C : P) ratio and of certain ‘essential’ fatty acids. These new hypotheses, the controversies they have provoked and the general lack of field studies prompted us to organize a workshop. Thus, the first international Plankton Ecology Group (PEG) workshop on The Role of Food Quality for Zooplankton was held from 17 to 21 March 1996 at the Centre of Limnology in Nieuwersluis, the Netherlands. The main objectives were to take stock of the state-of-the-art in food quality research, to address the factors that determine food quality, and to integrate the available information into a coherent and consistent view of food quality for the zooplankton. In contrast to most PEG meetings, we restricted the meeting to about 35 participants. However, international participation was broader than usual and included colleagues from North America and Asia, as well as Europe. The workshop’s organizers, Ramesh Gulati, a senior scientist, and Paul M. M. Weers, a post-doc at the Centre Of Limnology, approached potential speakers, in some cases suggesting topics for presentation. Each of five sessions was introduced with a review paper or a modelling paper.This special volume of Freshwater Biology contains twenty-six papers, including a summary by R. Gulati & B. DeMott. This paper briefly describes the findings of the workshop, and discusses them in light of the literature, discussions at the workshop and written comments from participants. Each of the workshop papers was peer reviewed, usually by two but in some cases by three referees. We learned that fatty acids and phosphorus are not mutually exclusive alternatives; the content of polyunsaturated acids of certain algae, especially eicosapentaenoic acid (EPA), is markedly affected by the algal P content. Thus, elemental limitation and fatty acid limitation may have additive or interacting effects. Although further research might modify this view, the accumulating evidence for P limitation seems stronger than for fatty acids limitation. However, despite laboratory studies that have clarified the potential mechanisms of food quality, the paucity of field studies prevents a consensus on the importance of specific mechanisms in nature. We also learned much from aquaculture studies, which have developed techniques for enriching zooplankton food with fatty acids and essential nutrients. We believe that the workshop proceedings will both contribute to our understanding and stimulate further research on food quality for zooplankton. Lastly, we take cognizance that cyanobacterial toxins are an important area of food quality research, which for practical reasons, did not receive our attention. Several presentations, however, revealed that cyanobacteria are also potentially poor food due to both higher C:P ratios and lower fatty acid content than many other algal species.Paul Weers’ efforts in organizing the workshop were very helpful. We acknowledge the facilities, funds and encouragement provided by staff and administration of the Centre of Limnology. We are also grateful to the participants for their cooperation and to the reviewers, many of whom were workshop participants.RAMESH D. GULATI BILL DE MOTT     

A rapid introduction to neurological biochemistry using <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Short, cost-effective teaching activities are a useful way of providing an integrated view on biological processes. Here we describe a brief, hands-on workshop that allows pre-university students to explore their understanding of a neurological pathway from its chemical bases to phenotype. The workshop effectively introduces the students to data collection and analysis in an enjoyable way and at an appropriate level, determined by an end of session feedback survey. The design of the workshop can be adapted and scaled to generate diverse sessions such as university teaching practicals or summer school training workshops.     

External Quality Assessment for Tuberculosis Diagnosis and Drug Resistance in the European Union: A Five Year Multicentre Implementation Study

BackgroundExternal quality assurance (EQA) systems are essential to ensure accurate diagnosis of TB and drug-resistant TB. The implementation of EQA through organising regular EQA rounds and identification of training needs is one of the key activities of the European TB reference laboratory network (ERLTB-Net). The aim of this study was to analyse the results of the EQA rounds in a systematic manner and to identify potential benefits as well as common problems encountered by the participants.MethodsThe ERLTB-Net developed seven EQA modules to test laboratories’ proficiency for TB detection and drug susceptibility testing using both conventional and rapid molecular tools. All National TB Reference laboratories in the European Union and European Economic Area (EU/EEA) Member States were invited to participate in the EQA scheme.ResultsA total of 32 National TB Reference laboratories participated in six EQA rounds conducted in 2010–2014. The participation rate ranged from 52.9% - 94.1% over different modules and rounds. Overall, laboratories demonstrated very good proficiency proving their ability to diagnose TB and drug-resistant TB with high accuracy in a timely manner. A small number of laboratories encountered problems with identification of specific Non-tuberculous Mycobacteria (NTMs) (N = 5) and drug susceptibility testing to Pyrazinamide, Amikacin, Capreomycin, and Ethambutol (N = 4).ConclusionsThe European TB Reference laboratories showed a steady and high level of performance in the six EQA rounds. A network such as ERLTB-Net can be instrumental in developing and implementing EQA and in establishing collaboration between laboratories to improve the diagnosis of TB in the EU/EEA.     

Evaluation of a simplified semi-quantitative protocol for the estimation of Vibrio vulnificus in bathing water using cellobiose-colistin agar: a collaborative study with 13 municipal food controlling units in Denmark

A simplified semi-quantitative method using pre-enrichment in alkaline peptone water supplemented with polymyxin B and plating onto cellobiose-colistin (CC) agar for the estimation of Vibrio vulnificus in bathing water was evaluated. This protocol was tested in a collaborative study with 13 food controlling laboratories in Denmark during the 1999 bathing season in periods when water temperatures exceeded 20 degrees C. The average percentage of yellow colonies larger than 1 mm in diameter on CC agar that could be identified as V. vulnificus by colony hybridization with a species-specific DNA probe was 79%. This high percentage of specificity demonstrated that by using CC agar in estimating the level of V. vulnificus in bathing water, recognition of yellow colonies larger than 1 mm is sufficient for the identification of V. vulnificus with no further characterization needed. The simplified protocol may be included in the routine control of the microbial quality of bathing water done by the local food controlling laboratories, since it involves simple traditional and low-cost microbiological methods with no use of molecular skills or sophisticated equipment.     

Microbes in food processing technology

Abstract: There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-haled methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.     

Teaching cardiac electrophysiology modeling to undergraduate students: laboratory exercises and GPU programming for the study of arrhythmias and spiral wave dynamics

As part of a 3-wk intersession workshop funded by a National Science Foundation Expeditions in Computing award, 15 undergraduate students from the City University of New York(1) collaborated on a study aimed at characterizing the voltage dynamics and arrhythmogenic behavior of cardiac cells for a broad range of physiologically relevant conditions using an in silico model. The primary goal of the workshop was to cultivate student interest in computational modeling and analysis of complex systems by introducing them through lectures and laboratory activities to current research in cardiac modeling and by engaging them in a hands-on research experience. The success of the workshop lay in the exposure of the students to active researchers and experts in their fields, the use of hands-on activities to communicate important concepts, active engagement of the students in research, and explanations of the significance of results as the students generated them. The workshop content addressed how spiral waves of electrical activity are initiated in the heart and how different parameter values affect the dynamics of these reentrant waves. Spiral waves are clinically associated with tachycardia, when the waves remain stable, and with fibrillation, when the waves exhibit breakup. All in silico experiments were conducted by simulating a mathematical model of cardiac cells on graphics processing units instead of the standard central processing units of desktop computers. This approach decreased the run time for each simulation to almost real time, thereby allowing the students to quickly analyze and characterize the simulated arrhythmias. Results from these simulations, as well as some of the background and methodology taught during the workshop, is presented in this article along with the programming code and the explanations of simulation results in an effort to allow other teachers and students to perform their own demonstrations, simulations, and studies.