Harmful and Potentially Harmful Constituents in E-Liquids and Aerosols from Electronic Nicotine Delivery Systems (ENDS).

In 2012, the U.S. Food & Drug Administration (FDA) published an established list of 93 harmful and potentially harmful constituents (HPHCs) targeting four tobacco product types (cigarettes, cigarette tobacco, roll-your-own tobacco, smokeless tobacco). In 2016, the FDA finalized the deeming rule to regulate electronic nicotine delivery systems (ENDS). However, knowledge gaps exist regarding whether certain HPHCs are present in ENDS e-liquids and aerosols. We identified and addressed these gaps by conducting literature searches and then experimentally quantifying HPHCs in the e-liquid and aerosol of 37 ENDS brands based on gaps in the literature. The literature searches identified 66 e-liquid HPHCs and 68 aerosol HPHCs that have limited to no information regarding the quantifiability of these constituents. A contracted ISO 17025 accredited laboratory performed the HPHC quantifications. The availability of validated analytical methods in the contracted laboratory determined the HPHCs included in the study scope (63/66 for e-liquids, 64/68 for aerosols). Combining the results from the quantifications and literature searches, 36 (39%) and 34 (37%) HPHCs were found quantifiable (≥limit of quantification [LOQ]) in ENDS e-liquids and aerosols, respectively, with 25 HPHCs being quantifiable in both matrices. Quantifiability results imply potential HPHC transfers between matrices, leaching from components, or formations from aerosol generation. The study results can inform the scientific basis for manufacturers and regulators regarding regulatory requirements for HPHC reporting. The HPHC quantities can also inform evaluations of the public health impact of ENDS and public communications regarding ENDS health risks.

Asthma heterogeneity among Asian American children: The California Health Interview Survey.

BACKGROUND: The Asian American (AsA) population is heterogenous and rapidly growing; however, little is known regarding childhood asthma burden among AsA ethnic groups. The relation between obesity and asthma in AsA ethnic groups also remains unclear. OBJECTIVE: To evaluate asthma prevalence and the relation of obesity to asthma risk among children in 7 AsA ethnic groups. METHODS: We analyzed data from the California Health Interview Survey from 2011 to 2020. AsA ethnicities were self-reported. Body mass index z-scores, calculated from self-reported height/weight, were used to categorize children by obesity status, based on body mass index-for-age growth charts. Prevalence of self-reported lifetime doctor-diagnosed asthma and asthma attack in the last 12 months was calculated. We performed multivariable logistic regressions adjusting for age and sex. RESULTS: Of 34,146 survey respondents, 12.2% non-Hispanic White and 12.5% AsA children reported lifetime asthma. Among AsA ethnic groups, however, lifetime asthma ranged from 5.1% (Korean American) to 21.5% (Filipino American). Non-Hispanic White children and AsA children had a similar lifetime asthma prevalence (adjusted odds ratio [aOR], 1.05; 95% CI, 0.71-1.55; P = .81), but prevalence was lower in Korean American children (aOR, 0.37; 95% CI, 0.19-0.73; P = .004) and higher in Filipino American children (aOR, 1.97; 95% CI, 1.22-3.17; P = .006). The lifetime asthma prevalence of different AsA ethnic groups persisted even when stratified by obesity status. CONCLUSION: Childhood lifetime asthma prevalence varied among AsA ethnic groups, with lowest prevalence in Korean American children and highest prevalence in Filipino American. Further characterization of asthma burden among AsA ethnic groups may help guide asthma screening and prevention measures and offer new insights into asthma pathogenesis.

Carbonyl Yields in Cigars under Three Smoking Regimens Using a Linear Smoking Machine.

This study used standard linear smoking machines and puffing protocols to generate data on carbonyl yields in mainstream smoke from 11 unfiltered sheet-wrapped cigars (SWC), seven leaf-wrapped cigars (LWC), and two Kentucky reference cigarettes (3R4F, 1R6F). Carbonyl yields in cigar and cigarette products were determined using three different smoking regimens: International Organization for Standardization (ISO), Canadian Intense (CI), and Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Recommended Method (CRM) No. 64 (CRM64, Routine Analytical Cigar-Smoking Machine─Specifications, Definitions and Standard Conditions). Mainstream tobacco smoke was collected using a smoking machine fitted with an impinger containing 2,4-dinitrophenylhydrazine (DNPH) and carbonyl compounds quantified using liquid chromatography with an ultraviolet detector. Commercial SWC and LWC generated comparable formaldehyde yields (SWC, 9.4-28 µg/cigar [ISO], 8.2-43 µg/cigar [CI], 8.6-13 µg/cigar [CRM64]; LWC, 11-13 µg/cigar [ISO], 11-22 µg/cigar [CI], 16-21 µg/cigar [CRM64]) and acrolein yields; however, LWC generated higher acetaldehyde yields compared to SWC, using CI and CRM64 regimens. Reference cigarettes using standard puffing regimens generated carbonyl yields within reported ranges and 5-10% RSDs, whereas the CRM64 regimen generated lower carbonyl yields and 12-14% RSDs. Reference cigarettes generated higher formaldehyde yields using cigarette smoking regimens (21-28 µg/cigarette under ISO, 76-96 µg/cigarette under CI) but comparable formaldehyde yields under CRM64 (12-14 µg/cigarette). In addition, this study evaluated physical parameters (e.g., tobacco weight, length, diameter, circumference, tobacco rod density) that show the correlation between tobacco weight, tobacco rod density, and acetaldehyde yields under the three smoking regimens. Carbonyl yields in the mainstream smoke of cigar products using the three smoking regimens were highly variable; however, the CI smoking regimen may provide meaningful analytical information regarding cigar smoke constituents, with lower likelihood of self-extinguishment due to the short puffing intervals.

Characterization of aspirin allergies in patients with coronary artery disease.

BACKGROUND: Aspirin prevents coronary thrombosis and is used extensively in cardiovascular prophylaxis. However, patients with a prior history of an aspirin "reaction" are routinely denied this medication. OBJECTIVE: To characterize the clinical presentation of a cohort of patients with coronary artery disease (CAD) and aspirin reactions. METHODS: Between 2009 and 2012, using a retrospective computer analysis, information was collected on all patients within a county-wide health care system presenting with CAD and a prior history of aspirin reactions. RESULTS: Of 9,565 patients with CAD, a prior history of aspirin reactions was recorded in 142 patients. Of these 142 patients, 30 (21%) had histories compatible with cutaneous and/or respiratory reactions. The other patients described adverse effects to aspirin, mostly gastrointestinal intolerance and bleeding. Aspirin-induced anaphylaxis was recorded in patients but may have been misdiagnosed, describing instead respiratory hypersensitivity reactions. Of the 142 patients, only 34 (24%) were receiving daily cardiovascular prophylaxis with aspirin. Of 108 patients not receiving aspirin, 25 (17.6%) were prescribed clopidogrel. CONCLUSION: Histories of aspirin reactions in patients with CAD are uncommon, occurring in only 1.5% of our study population. The 21% of patients with histories compatible with aspirin hypersensitivities can be challenged and, if the results are positive, successfully desensitized. Moreover, almost all patients with gastric intolerance to aspirin can be treated with aspirin and a proton pump inhibitor. However, both approaches, which result in restoration of cardiovascular prophylaxis, were seriously underused in our study population.

Harmful and Potentially Harmful Constituents in the Filler and Smoke of Tobacco-Containing Tobacco Products.

The U.S. Food and Drug Administration established a list of 93 harmful and potentially harmful constituents (HPHCs) in tobacco products. While HPHCs are required to be submitted for tobacco products, knowledge gaps exist regarding which tobacco-containing tobacco product (TCTP, i.e., tobacco products that contain tobacco(s) as a component) types (cigarettes, cigars, roll-your-own tobaccos [RYOs], pipe tobaccos [pipes], smokeless tobacco products [STPs], waterpipe tobaccos [waterpipes]) and matrices (filler, smoke) contain which HPHCs. This study identified and addressed such gaps by conducting literature searches and measuring the amount of HPHCs in TCTP types and matrices. First, literature searches, performed for cigarettes, RYOs, and STPs for publications up to 2014 and for cigars, pipes, and waterpipes for publications up to 2016, identified knowledge gaps for the 93 HPHCs (or 119 HPHCs if cresols [o-, m-, p-cresol] are counted as 3 and chlorinated dioxins/furans as 25) across TCTP types and matrices. Then, three ISO 17025 accredited laboratories including two subcontracted laboratories performed the HPHC quantifications. Inclusion of the HPHCs, TCTP types, and matrices in the study scope was also determined by the availability of validated analytical methods in each laboratory. Eleven (9%) HPHCs are quantifiable in all brands for all TCTP types and matrices, 33 (28%) HPHCs are not quantifiable in any brands of any TCTP type and matrix, and 74 (63%) HPHCs are quantifiable only in some brands across TCTP types and matrices examined. Understanding the quantifiability of HPHCs in each TCTP type and matrix can inform the scientific basis for manufacturers regarding the regulatory requirements for reporting HPHCs. The quantity of HPHCs observed can also inform the evaluation of the public health impact of HPHCs and public communications regarding the health risks of tobacco products.

Development and Validation Of A Discriminatory Dissolution Method for Portioned Moist Snuff and Snus.

Portioned moist snuff and snus, two subcategories of smokeless tobacco products (STP) were dissolution tested as a quality control test. A USP Apparatus 4 was employed to develop and validate the method. The method was assessed based on time to reach nicotine dissolution plateau, percentage difference between two profiles at each time point, relative standard deviation (RSD), and f1 (similarity) and f2 (dissimilarity) values. Based on these criteria, 200 ml volume and 8 ml/min flow were found be discriminatory. The amount of nicotine dissolved from the nine products varied widely (2.0-3.4, 2.1-4.1, 3.3-4.6, 5.5-6.6, 6.9-9.1, 11.5-14.2, 12.5-14.6, 14.0-15.5, and 15.5-19.6 mg/pouch at 60 min). RSDs of the dissolution ranges were more than 20% at earlier time points and less than 20% at later timepoints. The developed method produced distinct profiles for all the tested products, which was further confirmed by f1>15 and f2<50 values. In conclusion, the developed method was discriminatory and can be employed as a quality control test and to differentiate among moist snuff and snus products.

Bridging of a Liquid Chromatography-Tandem Mass Spectrometry Method for Oxytetracycline, Chlortetracycline, and Tetracycline in Bovine Kidney with the Official Microbial Growth Inhibition Assay.

BACKGROUND: Oxytetracycline (OTC), chlortetracycline (CTC), and tetracycline (TC) are approved antibiotics used to treat bacterial infections in cattle. To ensure human food safety, a tolerance has been established for the sum of these three TC residues as 12 parts per million in bovine kidney in the United States The current official regulatory method for quantifying these antibiotics in the target organ is a labor-intensive microbiological assay. OBJECTIVE: Our laboratory developed and validated a fast, selective, and less laborious method utilizing LC-tandem mass spectrometry for the determination and confirmation of the three tetracyclines (TET) in bovine kidney. METHODS: Briefly, homogenized kidney tissue was spiked with an internal standard (ISTD), and then was extracted with 1% phosphate buffer. The crude extract was cleaned up using solid-phase extraction cartridges before instrumental analysis. RESULTS: Accuracies for quantifying these three drugs in fortified kidney homogenate were between 99.9 and 110% at multiple concentrations, with respective CVs all below 9.5%. Quantitative correlation between the two methods (bridging) was evaluated with incurred bovine kidney samples for each of the three tetracyclines separately. The results were statistically evaluated using a measurement model called Functional Relationship Estimation by Maximum Likelihood. CONCLUSION: A linear quantitative relationship was demonstrated between the two methods within the concentration range of regulatory relevance. HIGHLIGHTS: This instrumental method is in addition to the established microbial assay for the detection of tetracyclines residue in beef kidney to ensure the food safety of cattle products.

Methicillin-resistant Staphylococcus aureus pelvic abscesses in a female after gynecologic pelvic surgery.

Pelvic abscesses occurring after gynecologic pelvic surgery are uncommon. We describe the case of a woman who, after undergoing such a procedure, was found to have pelvic abscesses infected with methicillin-resistant Staphyloccocus aureus. The purpose of this report is to raise awareness of a life-threatening complication of gynecologic pelvic surgery.

Identification and molecular cloning of Atlantic cod (Gadus morhua) activating transcription factor 3 (ATF3) transcript and its induction in spleen following intraperitoneal polyriboinosinic polyribocytidylic acid injection.

Activating transcription factor 3 (ATF3) participates in cellular processes to adapt to various extra- and intra-cellular changes including the modulation of immunity to prevent uncontrolled immune responses to pathogens. In teleost fishes, the involvement of ATF3 in immune response has not been documented. In this study, the putative Atlantic cod (Gadus morhua) ATF3 transcript was identified by performing rapid amplification of cDNA ends (RACE) based on unknown expressed sequence tags (ESTs) that are potentially inducible by polyriboinosinic polyribocytidylic acid (pIC, a synthetic double-stranded RNA viral mimic) in Atlantic cod. ATF3-like ESTs were the most abundant unknown transcript (i.e. lacking significant BLAST hits) generated from a previously constructed cDNA library enriched for pIC inducible transcripts in Atlantic cod spleen. The full-length cDNA of cod ATF3 consists of 2329 nucleotides with an open reading frame (ORF) of 735 bp encoding 244 amino acids. The deduced amino acid sequence of Atlantic cod ATF3 shares over 45% identity with its putative orthologs from other vertebrates. In addition, the presence of a conserved basic region leucine zipper (bZIP) domain in the deduced Atlantic cod ATF3-like protein further supports its identity as an ATF3 homolog. In the spleen of Atlantic cod challenged with intraperitoneal (IP) injections of pIC, the time-course transcript expression of ATF3 was studied using quantitative reverse transcription-polymerase chain reaction (QPCR). At 6 h following the pIC injection, the relative expression level of ATF3 mRNA was significantly up-regulated in comparison to a pre-injected control (61.9-fold) and its time-matched saline-injected control (97.3-fold). At 24 h following the pIC injection, the mRNA expression level of cod ATF3 had subsided and was no longer significantly different from its pre-injected control, but significantly higher (1.88-fold) than its time-matched saline-injected control. Collectively, these results suggest that ATF3 may be involved in the modulation of innate anti-viral response in Atlantic cod.

Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida.

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod (Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1 beta, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

Beyond Avoidance: the Psychosocial Impact of Food Allergies.

Over the past few years, the rates of food allergies have dramatically increased. As a result, the lives of patients and their caregivers have been dramatically altered. While most attention surrounding food allergies has focused on treatment, less consideration has been given to the mental health ramifications of living with this condition, among them depression, anxiety, post-traumatic stress, being bullied, and an overall poorer quality of life. At the same time, patients' family lives are often disrupted. Parents of food-allergic children, especially mothers, report anxiety, depression, and a decreased quality of life. Indeed, mental health issues associated with food allergies are likely underrecognized. In this review, we describe not only the psychosocial impacts of food allergies but also survey treatments that can be used to address this burgeoning problem. Interventions include educating members of the greater community about food allergies, camps for food allergic children, and support groups for parents. For physicians, treatment options consist of oral challenges, proximity challenges, oral immunotherapy, and cognitive behavioral therapy. Although the existing research is built on an already strong foundation, ultimately more studies are needed to deepen our understanding of the relationship between food allergies and mental health.

Histamine (Scombroid) Fish Poisoning: a Comprehensive Review.

Histamine fish poisoning, also known as scombroid poisoning, is the most common cause of ichythyotoxicosis worldwide and results from the ingestion of histamine-contaminated fish in the Scombroidae and Scomberesocidae families, including mackerel, bonito, albacore, and skipjack. This disease was first described in 1799 in Britain and re-emerged in the medical literature in the 1950s when outbreaks were reported in Japan. The symptoms associated with histamine fish poisoning are similar to that of an allergic reaction. In fact, such histamine-induced reactions are often misdiagnosed as IgE-mediated fish allergy. Indeed, histamine fish poisoning is still an underrecognized disease. In this review, we discuss the epidemiology, pathophysiology, evaluation, and treatment of scombroid disease. Because more than 80% of fish consumed in the USA is now imported from other countries, the disease is intimately linked with the global fish trade (National Marine Fisheries Service, 2012). Preventing future scombroid outbreaks will require that fishermen, public health officials, restaurant workers, and medical professionals work together to devise international safety standards and increase awareness of the disease. The implications of scombroid poisoning go far beyond that of fish and have broader implications for the important issues of food safety.

Characterization of the fatty acyl elongase (elovl) gene family, and hepatic elovl and delta-6 fatty acyl desaturase transcript expression and fatty acid responses to diets containing camelina oil in Atlantic cod (Gadus morhua).

For aquaculture to become sustainable, there is a need to substitute fish oil [FO, rich in ω3 long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5ω3 (EPA) and 22:6ω3 (DHA)] in aquafeed with plant oils such as camelina oil [CO, rich in C18 PUFA such as 18:3ω3 (ALA) and 18:2ω6 (LNA)]. The LC-PUFA are essential components in fish diets for maintaining optimal health, physiology and growth. However, most marine fish including Atlantic cod are inefficient at producing LC-PUFA from shorter chain precursors. Since elovl genes encode enzymes that play key roles in fatty acid biosynthesis, we hypothesized that they may be involved in Atlantic cod responses to diets rich in 18:3ω3 and 18:2ω6. Ten members of the cod elovl gene family were characterized at the mRNA level. RT-PCR was used to study constitutive expression of elovl transcripts in fifteen tissues. Some transcripts (e.g. elovl5) were ubiquitously expressed, while others had tissue-specific expression (e.g. elovl4a in brain and eye). Cod fed a CO-containing diet (100% CO replacement of FO and including solvent-extracted fish meal) had significantly lower weight gain, with significant up-regulation of elovl5 and fadsd6 transcripts in the liver as shown by QPCR analysis, compared with cod on a FO control diet after a 13-week trial. Multivariate statistical analyses (SIMPER and PCA) indicated that high 18:3ω3 and/or low ω3 LC-PUFA levels in the liver were associated with the up-regulation of elovl5 and fadsd6, which are involved in LC-PUFA biosynthesis in cod.

Family-specific differences in growth rate and hepatic gene expression in juvenile triploid growth hormone (GH) transgenic Atlantic salmon (Salmo salar).

Growth hormone transgenic (GHTg) Atlantic salmon (Salmo salar) have enhanced growth when compared to their non-transgenic counterparts, and this trait can be beneficial for aquaculture production. Biological confinement of GHTg Atlantic salmon may be achieved through the induction of triploidy (3N). The growth rates of triploid GH transgenic (3NGHTg) Atlantic salmon juveniles were found to significantly vary between families in the AquaBounty breeding program. In order to characterize gene expression associated with enhanced growth in juvenile 3NGHTg Atlantic salmon, a functional genomics approach (32K cDNA microarray hybridizations followed by QPCR) was used to identify and validate liver transcripts that were differentially expressed between two fast-growing 3NGHTg Atlantic salmon families (AS11, AS26) and a slow-growing 3NGHTg Atlantic salmon family (AS25); juvenile growth rate was evaluated over a 45-day period. Of 687 microarray-identified differentially expressed features, 143 (116 more highly expressed in fast-growing and 27 more highly expressed in slow-growing juveniles) were identified in the AS11 vs. AS25 microarray study, while 544 (442 more highly expressed in fast-growing and 102 more highly expressed in slow-growing juveniles) were identified in the AS26 vs. AS25 microarray study. Forty microarray features (39 putatively associated with fast growth and 1 putatively associated with slow growth) were present in both microarray experiment gene lists. The expression levels of 15 microarray-identified transcripts were studied using QPCR with individual RNA samples to validate microarray results and to study biological variability of transcript expression. The QPCR results agreed with the microarray results for 12 of 13 putative fast-growth associated transcripts, but QPCR did not validate the microarray results for 2 putative slow-growth associated transcripts. Many of the 39 microarray-identified genes putatively associated at the transcript expression level with fast-growing 3NGHTg salmon juveniles (including APOA1, APOA4, B2M, FADSD6, FTM, and GAPDH) are involved in metabolism, iron homeostasis and oxygen transport, and immune- or stress-related responses. The results of this study increase our knowledge of family-specific impacts on growth rate and hepatic gene expression in juvenile 3NGHTg Atlantic salmon. In addition, this study provides a suite of putative rapid growth rate-associated transcripts that may contribute to the development of molecular markers [e.g. intronic, exonic or regulatory region single nucleotide polymorphisms (SNPs)] for the selection of GHTg Atlantic salmon broodstock that can be utilized to produce sterile triploids of desired growth performance for future commercial applications.

The united allergic airway: connections between allergic rhinitis, asthma, and chronic sinusitis.

BACKGROUND: The united allergic airway is a theory that connects allergic rhinitis (AR), chronic rhinosinusitis, and asthma, in which seemingly disparate diseases, instead of being thought of separately, are instead viewed as arising from a common atopic entity. OBJECTIVE: This article describes patients with such diseases; explores ideas suggesting a unified pathogenesis; elucidates the various treatment modalities available, emphasizing nasal corticosteroids and antihistamines; and provides an update of the literature. METHODS: A literature review was conducted. CONCLUSION: The aggregation of research suggests that AR, asthma, and chronic rhinosinusitis are linked by the united allergic airway, a notion that encompasses commonalities in pathophysiology, epidemiology, and treatment.

Characterization and expression studies of Gaduscidin-1 and Gaduscidin-2; paralogous antimicrobial peptide-like transcripts from Atlantic cod (Gadus morhua).

Piscidins are a family of antimicrobial peptides (AMPs) from fish that constitute an important component of their innate immune system. Based on previously generated Atlantic cod (Gadus morhua) expressed sequence tags (ESTs), we identified sequences representing two paralogous AMP-like transcripts. These Atlantic cod paralogues were termed gaduscidins (GAD-1 and GAD-2), derived from the genus name Gadus. We obtained full-length cDNA sequences for these putative AMP-encoding transcripts using bi-directional rapid amplification of cDNA ends (RACE). GAD-1 and GAD-2 putative peptides exhibit sequence similarity with members of the piscidin family from teleost fish. Quantitative reverse transcription - polymerase chain reaction (QPCR) was utilized in transcript expression studies of GAD-1 and GAD-2. We examined the constitutive expression of these transcripts in six tissues (head kidney, blood, brain, gill, pyloric caecum, and spleen) of non-stressed juvenile cod; GAD-1 and GAD-2 transcripts were detected in all six tissues, with the highest expression of both transcripts being in spleen, head kidney, and gill. Transcript expression of GAD-1 and GAD-2 was also examined in immune tissues (spleen and head kidney) following intraperitoneal (IP) injection of formalin-killed, atypical Aeromonas salmonicida (ASAL) or phosphate-buffered saline (PBS control). Both transcripts were weakly (less than 4-fold) up-regulated by ASAL in spleen but non-responsive to ASAL in head kidney. Since GAD-1 and GAD-2 transcripts are highly expressed constitutively in immune-relevant tissues (e.g. spleen and head kidney), and are weakly induced in spleen following IP injection with bacterial antigens, they may represent important components of innate immunity in Atlantic cod.

An integrated approach to gene discovery and marker development in Atlantic cod (Gadus morhua).

Atlantic cod is a species that has been overexploited by the capture fishery. Programs to domesticate this species are underway in several countries, including Canada, to provide an alternative route for production. Selective breeding programs have been successfully applied in the domestication of other species, with genomics-based approaches used to augment conventional methods of animal production in recent years. Genomics tools, such as gene sequences and sets of variable markers, also have the potential to enhance and accelerate selective breeding programs in aquaculture, and to provide better monitoring tools to ensure that wild cod populations are well managed. We describe the generation of significant genomics resources for Atlantic cod through an integrated genomics/selective breeding approach. These include 158,877 expressed sequence tags (ESTs), a set of annotated putative transcripts and several thousand single nucleotide polymorphism markers that were developed from, and have been shown to be highly variable in, fish enrolled in two selective breeding programs. Our EST collection was generated from various tissues and life cycle stages. In some cases, tissues from which libraries were generated were isolated from fish exposed to stressors, including elevated temperature, or antigen stimulation (bacterial and viral) to enrich for transcripts that are involved in these response pathways. The genomics resources described here support the developing aquaculture industry, enabling the application of molecular markers within selective breeding programs. Marker sets should also find widespread application in fisheries management.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.