Collection of Data on Pesticides in Maize and Tomato in Africa: Protocol for Africa Pesticide Residue Survey Study.

Pesticide use has grown rapidly in West Africa over the past decades. Regulatory capacity has not kept pace with the rapid proliferation of pesticide products and on-farm use. As a result, health and environmental impacts from the growing use of pesticides, despite their potential importance to food safety, remain largely unmonitored, underreported, and poorly understood by key stakeholders. This study protocol was the document for conducting a pesticide survey study to identify the most critically emerging pesticides across the Continent of Africa. Multiple countries were selected in this study to represent the north, east, south, and west regions of Africa. Two food commodities, maize and tomato, were chosen to monitor the pesticide level for food safety. This study protocol describes the fieldwork and laboratory work per the standards of Good Laboratory Practices (GLP) and ISO-17025 and US EPA 860 Residue Chemistry Guidelines but the survey study performed was not considered as a GLP or ISO 17025 study. This is because many steps were not able to be closely monitored per the GLP requirements. This protocol describes the requirements for a pesticide residue study in food collected from local markets. This protocol describes the test commodities, sampling methods, sample transfer/shipping, storage stability, sample analysis, sample disposal, and documentation and record keeping.

Prevalences of Shiga toxin subtypes and selected other virulence factors among Shiga-toxigenic Escherichia coli strains isolated from fresh produce.

Shiga-toxigenic Escherichia coli (STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have the eae gene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among the eae-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. The ehxA gene, which encodes enterohemolysin, was found in ∼60% of the isolates, and the saa and subAB genes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. The stx1a and stx2a subtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. The stx2d subtype was the second most common subtype (28% overall), followed by stx2c (7.5%), and only 2 to 3% of the produce STEC strains had the stx2e and stx2g subtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.

Prevalence and Characteristics of Salmonella Serotypes Isolated from Fresh Produce Marketed in the United States.

Salmonella continues to rank as one of the most costly foodborne pathogens, and more illnesses are now associated with the consumption of fresh produce. The U.S. Department of Agriculture Microbiological Data Program (MDP) sampled select commodities of fresh fruit and vegetables and tested them for Salmonella, pathogenic Escherichia coli, and Listeria. The Salmonella strains isolated were further characterized by serotype, antimicrobial resistance, and pulsed-field gel electrophoresis profile. This article summarizes the Salmonella data collected by the MDP between 2002 and 2012. The results show that the rates of Salmonella prevalence ranged from absent to 0.34% in cilantro. A total of 152 isolates consisting of over 50 different serotypes were isolated from the various produce types, and the top five were Salmonella enterica serotype Cubana, S. enterica subspecies arizonae (subsp. IIIa) and diarizonae (subsp. IIIb), and S. enterica serotypes Newport, Javiana, and Infantis. Among these, Salmonella serotypes Newport and Javiana are also listed among the top five Salmonella serotypes that caused most foodborne outbreaks. Other serotypes that are frequent causes of infection, such as S. enterica serotypes Typhimurium and Enteritidis, were also found in fresh produce but were not prevalent. About 25% of the MDP samples were imported produce, including 65% of green onions, 44% of tomatoes, 42% of hot peppers, and 41% of cantaloupes. However, imported produce did not show higher numbers of Salmonella-positive samples, and in some products, like cilantro, all of the Salmonella isolates were from domestic samples. About 6.5% of the Salmonella isolates were resistant to the antimicrobial compounds tested, but no single commodity or serotype was found to be the most common carrier of resistant strains or of resistance. The pulsed-field gel electrophoresis profiles of the produce isolates showed similarities with Salmonella isolates from meat samples and from outbreaks, but there were also profile diversities among the strains within some serotypes, like Salmonella Newport.

Methods for gene expression profiling in dermatology research using DermArray nylon filter DNA microarrays.

Here we present methods of gene expression profiling using nylon filter deoxyribonucleic acid (DNA) microarrays and radiolabeled and nonradiolabeled hybridization probes. DermArray(R) nylon filter DNA microarrays were designed specifically for use in dermatology research. A patent-pending method was used to select approx 4400 highly informative, sequence-verified human cDNA clones for this DNA micro array. Using DermArray(R) filters, biomarkers have been discovered for normal and pathologic cells from skin, and for responses to dermatologic drugs. As an example, gene expression profiling was performed with hydroquinone-treated SKMel-28 cells, a melanoma cell line. Also included are the methods for bioinformatic analysis using Pathwaystrade mark software.

Regulation of gene expression in inflammatory bowel disease and correlation with IBD drugs: screening by DNA microarrays.

Potential biomarkers for Crohn's disease (CD) and ulcerative colitis (UC) were identified from two sets of full thickness pathologic samples utilizing DermArray and PharmArray DNA microarrays relative to uninvolved (Un) colon or normal colon. Seven of the over-expressed genes were verified using quantitative RT-PCR (i.e., TMPT, FABP1, IFI27, LCN2, COL11A2, HXB, and metallothionein). By correlating gene expression profiles between inflammatory bowel disease (IBD) tissue samples and IBD drug-treated cell cultures it might be possible to identify new candidate molecular target genes for IBD therapy and drug discovery. Potential biomarkers for CaCo2 cell cultures, which are routinely used as a GI tract surrogate model for in vitro pharmacokinetic studies, treated with azathioprine, 5-aminosalicylic acid, metronidazole, and prednisone were also identified from another experiment. Metallothionein mRNA expression was found to be down-regulated in azathioprine-treated CaCo2 cells, and was coincidentally up-regulated in the CD sample, thus resulting in an anti-correlation. These results suggest that this new screening methodology is feasible, that metallothioneins might be biomarkers for azathioprine therapy in vivo in CD, and that azathioprine might mechanistically down-regulate metallothionein gene expression. Correlations were also observed between IBD samples and either metronidazole- or 5-aminosalicylic acid-treated CaCo2 cells. Similar comparisons of disease tissue samples in vivo vs drug-treated cell cultures in vitro might reveal new mechanistic insights concerning established or experimental drug therapies. This affordable in vitro methodology is promising for expanded studies of IBD and other diseases.

Prevalence and diversity of enterotoxigenic Escherichia coli strains in fresh produce.

Analysis of fresh produce showed that enterotoxigenic Escherichia coli (ETEC) strains are most often found in cilantro and parsley, with prevalence rates of approximately 0.3%. Some ETEC strains also carried Shiga toxigenic E. coli (STEC) genes but had no STEC adherence factors, which are essential to cause severe human illness. Most ETEC strains in produce carried stable toxin and/or labile toxin genes but belonged to unremarkable serotypes that have not been reported to have caused human illnesses.

Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR.

Squamous cell carcinoma (SCC) is the second most common form of skin cancer in Caucasians. Here we report on the identification of biomarkers of human cutaneous SCC cell lines in vitro and tissue samples in vivo using DermArray and PharmArray DNA microarrays, consisting of ca. 7400 unique human cDNAs. Differentially expressed genes were identified in two facial skin SCC cell lines (SCC 12 and SCC 13) compared to normal keratinocytes, and three cutaneous SCC tissue samples compared to normal skin. Quantitative validations of up- and down-regulated biomarkers were performed by qRT-PCR on 23 biomarker genes for the cell lines and 20 biomarker genes for the tumor tissues. In addition, three oral SCC cell lines were also included in the qRT-PCR validations for comparison, and the biomarker profiles were highly similar between the cutaneous and the oral SCC cell lines for all 23 biomarkers examined. The expression profiles for a variety of non-cutaneous SCC types, such as head-and-neck, oral, and lung, have been previously published. This report is the first to describe biomarkers for cutaneous SCC in two contexts, in vitro and in vivo. Although there was minimal overlap between the two different contexts using DNA microarrays, five genes were found common to both the cell lines and tissues, namely fibronectin 1, annexin A5, glyceraldehyde 3-phosphate dehydrogenase, zinc-finger protein 254, and huntingtin-associated protein interacting protein. Some of our previously published biomarkers of normal keratinocytes were down-regulated in SCC, suggestive of the dedifferentiated status of the transformed cells. While recent reports have identified some of the same genes as SCC biomarkers, for instance in head-and-neck cancer, thereby validating our approach, we have identified some novel biomarkers for cutaneous disease. These biomarker lists may be useful in molecular diagnostics of non-melanoma skin cancer, and a subset of the biomarkers might serve as suitable targets for drug discovery efforts of therapies for SCC.

A method to improve selection of molecular targets by circumventing the ADME pharmacokinetic system utilizing PharmArray DNA microarrays.

DNA microarrays may be used to identify potential molecular targets for drug discovery. Yet, DNA microarray experiments provide massive amounts of data. To limit the choice of potential molecular targets, it may be desirable to eliminate genes coincidentally up-regulated in tissues implicated in absorption, distribution, metabolism, and excretion (ADME) pharmacokinetics. DNA microarray experiments were performed to demonstrate a gene-exclusion approach using as an example RNA samples of neural origin, i.e., a human neuroblastoma cell line (SK-N-SH) and brain tissue, as the intended hypothetical site(s) of drug action. Biomarkers were identified using PharmArray DNA microarrays. The lists of neuroblastoma and neural biomarkers were constrained by limiting selection to the subset of genes that were not highly expressed in three transformed cell lines from liver, colon, and kidney (HepG2, Caco-2, and 786-O, respectively) that are routinely used as representatives of the ADME system during in vitro pharmacology and toxicology experiments. Principal component analysis methods with likelihood ratio-related bioinformatic tools were utilized to identify robust potential biomarker genes for the three ADME-related cell lines, neuroblastoma, and normal brain. Biomarkers of each sample were identified and selected genes were validated by qRT-PCR. Hundreds of biomarkers of the three ADME-related cell types, representing hepatocytes, kidney epithelium, and gastrointestinal tract, may now be used as a valuable database to restrict selection of biomarkers as potential molecular targets from the intended samples (e.g., neuroblastoma in this work). In addition to biomarker discovery per se, this demonstration suggests that our model method may be viable to help restrict gene lists during selection of potential molecular targets for subsequent drug discovery.