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Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses.

Omitted variable bias occurs in most statistical models. Whenever a confounding variable that is correlated with both dependent and independent variables is omitted from a statistical model, estimated effects of included variables are likely to be biased due to omitted variables. This issue is particularly problematic in physics education research where many research studies are quasiexperimental or observational in nature due to ethical and logistical limitations. In this paper, we illustrate the mechanisms behind omitted variable bias in explanatory modeling using authentic data and analytical solutions. We demonstrate that omitting confounding variables that are strongly correlated with included variables and have large effects on the dependent variable can significantly bias estimated effects for included variables. We also find that controlling for variables that are uncorrelated with other variables or have no effect on the dependent variable does not appreciably bias estimated effects and may or may not affect the precision of those estimates. These results suggest that removing from explanatory models variables that are not “statistically significant” can have unintended consequences on model and variable interpretations. Our results underscore the importance of carefully considering why or why not to include a variable in a model, informed by both data and theory.

Background: The genetic causes of atrial fibrillation (AF) with slow conduction are unknown. Methods: Eight kindreds with familial AF and slow conduction, including a family affected by early onset AF, heart block and incompletely penetrant non-ischemic cardiomyopathy (NICM) underwent whole exome sequencing. Results: A known pathogenic mutation in the desmin (DES) gene resulting in S13F substitution at a PKC phosphorylation site was identified in all four members of the kindred with early-onset AF and heart block, while only two developed NICM. Higher penetrance of the mutation for AF and heart block prompted the screening for DES modifier(s). A second deleterious mutation in the phosphodiesterase 4D interacting-protein (PDE4DIP) gene resulting in A123T substitution segregated with early onset AF, heart block and the DES mutation. Three additional novel deleterious PDE4DIP mutations were identified in four other unrelated kindreds. Characterization of PDE4DIP in vitro suggested impaired compartmentalization of PKA and PDE4D characterized by reduced colocalization with PDE4D, increased cAMP activation leading to higher PKA phosphorylation of the β2-adrenergic-receptor, and decreased PKA phosphorylation of Desmin in response to isoproterenol stimulation compared to wildtype PDE4DIP. Conclusion: Our findings identify an epistatic interaction between DES and PDE4DIP variants, increasing the penetrance for conduction disease and arrhythmia.

Undergraduate labs are more effective and more positive for students if they encourage investigation and decision-making, not verification of textbook concepts.

Instructional labs are being transformed to better reflect authentic scientific practice, often by removing aspects of pedagogical structure to support student agency and decision making. We explored how these changes impact men’s and women’s participation in group work associated with labs through clustering methods on the quantified behavior of students. We compared the group roles students take on in two different types of instructional settings: (i) highly structured traditional labs, and (ii) less structured inquiry-based labs. Students working in groups in the inquiry-based (less structured) labs assumed different roles within their groups, however men and women systematically took on different roles and men behaved differently when in single- versus mixed-gender groups. We found no such systematic differences in role division among male and female students in the traditional (highly structured) labs. Students in the inquiry-based labs were not overtly assigned these roles, indicating that the inequitable division of roles was not a result of explicit assignment. Our results highlight the importance of structuring equitable group dynamics in educational settings, as a gendered division of roles can emerge without active intervention. As the culture in physics evolves to remove systematic gender biases in the field, instructors in educational settings must not only remove explicitly biased aspects of curricula but also take active steps to ensure that potentially discriminatory aspects are not inadvertently reinforced.

Many institutions are changing the focus of their introductory physics labs from verifying physics content towards teaching students about the skills and nature of science. As instruction shifts, so too will the ways students approach and behave in the labs. In this study, we evaluated students' lab notes from an early activity in an experimentation-focused lab course. We found that about 30% of student groups (out of 107 groups at three institutions) recorded questionable research practices in their lab notes, such as subjective interpretations of results or manipulating equipment and data. The large majority of these practices were associated with confirmatory goals, which we suspect stem from students' prior exposure to verification labs. We propose ways for experimentation-focused labs to better engage students in the responsible conduct of research and authentic scientific practice., Comment: 4 pages, 4 figures

While laboratory instruction is a cornerstone of physics education, the impact of student behaviours in labs on retention, persistence in the field, and the formation of students' physics identity remains an open question. In this study, we performed in-lab observations of student actions over two semesters in two pedagogically different sections of the same introductory physics course. We used a cluster analysis to identify different categories of student behaviour and analyzed how they correlate with lab structure and gender. We find that, in lab structures which fostered collaborative group work and promoted decision making, there was a task division along gender lines with respect to laptop and equipment usage (and found no such divide among students in guided verification labs)., Comment: 4 pages, 3 figures, 3 tables

Nonsyndromic patent ductus arteriosus (PDA) is a common congenital heart defect (CHD) with both inherited and acquired causes, but the disease mechanisms have remained elusive. Using combined genome-wide linkage analysis and whole-exome sequencing (WES), we identified independent mutations in PRDM6, which encodes a nuclear protein that is specific to vascular smooth muscle cells (VSMC), has histone methyl transferase activities, and acts as a transcriptional suppressor of contractile proteins. In vitro assays showed that the mutations cause loss of function either by intracellular redistribution of the protein and/or by alteration of its methyltransferase activities. Wild-type embryonic ductus arteriosus (DA) exhibited high levels of PRDM6, which rapidly declined postnatally as the number of VSMCs necessary for ductus contraction increased. This dynamic change suggests that PRDM6 plays a key role in maintaining VSMCs in an undifferentiated stage in order to promote their proliferation and that its loss of activity results in premature differentiation and impaired remodeling of the DA. Our findings identify PRDM6 mutations as underlying genetic causes of nonsyndromic isolated PDA in humans and implicates the wild-type protein in epigenetic regulation of ductus remodeling.

Radio pulsar B1933+16 is brightest core-radiation dominated pulsar in the Arecibo sky, and here we carry out a comprehensive high resolution polarimetric study of its radiation at both 1.5 and 4.6 GHz. At 1.5 GHz, the polarization is largely compatible with a rotating-vector model with $\alpha$ and $\beta$ values of 125 and --1.2$^{\circ}$, such that the core and conal regions can be identified with the primary and secondary polarization modes and plausibly with the extraordinary and ordinary propagation modes. Polarization modal segregation of profiles shows that the core is comprised of two parts which we associate with later X-mode and earlier O-mode emission. Analysis of the broad microstructures under the core shows that they have similar timescales to those of the largely conal radiation of other pulsars studied earlier. Aberration/retardation analysis was here possible for both the conal and core radiation and showed average physical emission heights of about 200 km for each. Comparison with other core-cone pulsars suggests that the core and conal emission arises from similar heights. Assuming the inner vacuum gap model, we note that at these emission altitudes the frequency of the observed radiation $\nu_{obs}$ is less than the plasma frequency $\nu_p$. We then conclude that the radio emission properties are consistent with the theory of coherent curvature radiation by charged solitons where the condition $\nu_{obs} < \nu_{p}$ is satisfied. However, the differences that exist between core and conal emission in their geometric locations within a pulse, polarization and modulation properties are yet to be understood., Comment: 14 pages, 8 figures, 3 tables, accepted for publication in MNRAS

Recovery of functional beta cell mass offers a biological cure for type 1 diabetes. However, beta cell mass is difficult to regain once lost since the proliferation rate of beta cells after youth is very low. Angiopoietin like-protein 8 (ANGPTL8), a peptide that has a role in the regulation of lipoprotein lipase activity, was reported to increase beta cell proliferation in mice in 2013. Subsequent studies of human ANGPTL8 for short term (3 to 8 days) in non-diabetic mice showed little or no increase in beta cell proliferation. Here, we examined the effect of ANGPTL8 on glucose homeostasis in models that have not been examined previously. We expressed mouse ANGPTL8 using adenovirus in 2 mouse models of diabetes (streptozotocin and Non-Obese Diabetic (NOD) mice) over 2 weeks. Also, we tested ANGPTL8 in NOD mice deficient in leukocyte 12-lipoxygenase (12LO), an enzyme that contributes to insulitis and loss of beta cell function in NOD, in an effort to determine whether 12LO deficiency alters the response to ANGPTL8. Adenovirus-mediated expression of ANGPTL8 lowered blood glucose levels in streptozotocin treated mice without an increase in beta cell proliferation or serum insulin concentration. While ANGPTL8 did not reverse hyperglycemia in overtly hyperglycemic NOD mice or alter glucose homeostasis of non-diabetic NOD mice, ANGPTL8 reduced blood glucose levels in 12LOKO NOD mice. However, the lower glucose levels in 12LOKO NOD were not associated with higher serum insulin levels or beta cell proliferation. In summary, while mouse ANGPTL8 does not increase beta cell proliferation in NOD mice or streptozotocin treated mice in agreement with studies in non-diabetic mice, it lowers blood glucose levels in multiple low-dose streptozotocin induced diabetes and 12LO deficiency indicating that host factors influence the impact of ANGPTL8 on glucose homeostasis.

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.

Calls for reform to instructional labs means many instructors and departments are facing the daunting task of identifying goals for their introductory lab courses. Fortunately, the American Association of Physics Teachers (AAPT) released a set of recommendations for learning goals for the lab to support lab redevelopment [1]. Here we outline the process we have undergone to identify a set of learning goals for the labs that operationalize those provided by the AAPT. We also provide two examples of newly developed lab activities that aim to meet those goals to demonstrate this operationalization. We aim to provide departments and instructors with a few ideas of a procedure that they can follow or a set of goals that they can use to align lab instruction with the AAPT learning goals., 6 pages, 4 figures, accepted to The Physics Teacher

Standard audiometric evaluations are not sensitive enough to identify hidden hearing loss (HHL) and/or cochlear synaptopathy (CS). Patients with either of these conditions frequently present with difficulty understanding speech in noise or other complaints such as tinnitus. The purpose of this systematic review is to identify articles in peer-reviewed journals that assessed the sensitivity of audiologic measures for detecting HHL and/or CS, and which showed potential for use in a clinical test battery for these disorders. A reference librarian submitted specific boolean terminology to MEDLINE, Embase, and Web of Science. The authors used a consensus approach with specially designed score sheets for the selection of titles, abstracts, and then articles for inclusion in the systematic review and for quality assessment. Fifteen articles were included in the systematic review. Seven articles involved humans; seven involved animals, and one study used both humans and animals. Results showed that pure-tone audiometry to 20 kHz, otoacoustic emissions, electrocochleography, auditory brainstem response (ABR), electrophysiological tests, speech recognition in noise with and without temporal distortion, interviews, and self-report measures have been used to assess HHL and/or CS. For HHL, ultra-high-frequency audiometry may help identify persons with sensory hair cell loss that does not show up on standard audiograms. Promising nonbehavioral measures for CS included ABR wave I amplitude, the summating potential-to-action potential ratio, and speech recognition in noise with and without temporal distortion. Self-report questionnaires also may help identify auditory dysfunction in persons with normal hearing.

Framing affects how students interpret, approach, and accomplish tasks. Little is known, however, about how students frame tasks in physics labs. During the first lab of a sequence designed to teach students about modeling and critical thinking with data, students test a simple model of a pendulum that breaks down with improved measurements. Using in-lab video and follow-up interviews, we identified students' frequent use of a model-verifying frame that substantially interferes with the instructional goals. We present a case study analysis of two students who approach the lab with a model-verifying frame, engage in problematic behaviors including questionable research practices, but later shift their frames to accommodate goals aligned with instructional intention. As instructors transition their instructional labs to open-inquiry experiences, an activity that directly challenges the model-verifying frame may be productive for shifting students away from this problematic frame to one that supports their engagement in authentic experimentation.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance.

A countermeasure that protects the brain from organophosphate toxicity is an unmet need. Few small molecule reactivators that can cross the blood brain barrier and reactivate brain acetyl cholinesterases have been reported. Herein, we describe preclinical investigations of a new class of amidine-oxime reactivator of cholinesterases with improved potency and blood brain barrier permeability. (Z)-N-((E)-1-(Dimethylamino)-2-(hydroxyimino)ethylidene)butan-1-aminium chloride, 1, is zwitterionic at physiological pH but possesses increased oxime nucleophilicity because of the adjacent amidine functionality. The amidine-oximes reported herein were observed to be nontoxic (up to 200 mg/kg in vivo) and are chemically and metabolically stable. The results presented herein show that uncharged amidine-oxime reactivators such as 1 can penetrate the blood brain barrier in animals and protect from the toxicity of nerve agent model compounds.

Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.

Deficiency in complement component C1q is associated with an inability to clear apoptotic cells (efferocytosis) and aberrant inflammation in lupus, and identification of the pathways involved in these processes should reveal important regulatory mechanisms in lupus and other autoimmune or inflammatory diseases. In this study, C1q-dependent regulation of TNFα/IL-6 expression and efferocytosis was investigated using primary mouse bone marrow derived macrophages, and human monocyte derived macrophages. C1q downregulated LPS-dependent TNFα production in mouse and human macrophages. While prolonged stimulation with C1q (18 hours) was required to elicit a dampening of TNFα production from mouse macrophages, the human macrophages responded to C1q with immediate downregulation of TNFα. IL-6 production was unchanged in mouse and upregulated by human macrophages following prolonged stimulation with C1q. Our previous studies indicated that C1q programmed enhanced efferocytosis in mouse macrophages by enhancing expression of Mer tyrosine kinase and its ligand Gas6, a receptor-ligand pair that also inhibits proinflammatory signaling. Here we demonstrated that C1q-dependent programming of human macrophage efferocytosis required protein synthesis, however neither Mer nor the related receptor Axl were upregulated in human cells. In addition, while the C1q-collagen-like tails are sufficient for promoting C1q-dependent phagocytosis of antibody coated targets, the C1q-tails failed to program enhanced efferocytosis or dampen TNFα production. These data further elucidate the mechanisms by which C1q regulates proinflammatory signaling and efferocytosis in macrophages, functions that are likely to influence the progression of autoimmunity and chronic inflammation.

Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been well established in fish yet these studies are important for several reasons. First, such studies will provide functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnostic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic environment and adverse effects associated with drug–drug interactions, including CYP inhibition by pharmaceuticals may be of concern. Using liver microsomes from untreated and β-naphthoflavone (BNF)-treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish. These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the patterns seen here are different than what would be expected based on CYP inhibition in mammals.

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.

The ray-finned fishes make up about half of all vertebrate diversity and are by far the best represented group in the Animal Genome Size Database. However, they have traditionally been the least well investigated among vertebrates in terms of patterns and consequences of genome size diversity. This article synthesizes and expands upon existing information about genome size diversity in ray-finned fishes. Specifically, compiled data from the Animal Genome Size Database and FishBase are used to examine the potential patterns of interspecific genome size variability according to ecology, environment, morphology, growth, physiology, reproduction, longevity, and taxonomic diversity. Polyploidy and haploid genome sizes are considered separately, revealing differences in their respective consequences. This represents the most comprehensive summary of fish genome size diversity presented to date, and highlights areas of particular interest to investigate as more data become available.

Partial derivatives are used in a variety of different ways within physics. Most notably, thermodynamics uses partial derivatives in ways that students often find confusing. As part of a collaboration with mathematics faculty, we are at the beginning of a study of the teaching of partial derivatives, a goal of better aligning the teaching of multivariable calculus with the needs of students in STEM disciplines. As a part of this project, we have performed a pilot study of expert understanding of partial derivatives across three disciplines: physics, engineering and mathematics. Our interviews made use of the Partial Derivative Machine (PDM), which is a mechanical system featuring four observable and controllable properties, of which any two are independent. Using the PDM, we probed expert understanding of partial derivatives in an experimental context in which there is not a known functional form. Through these three interviews, we found that the mathematicians exhibited a striking difference in their understanding of derivatives relative to the other groups. The physicists and engineers were quick to use measurements to find a numeric approximation for a derivative. In contrast, the mathematicians repeatedly returned to speculation as to the functional form, and although they were comfortable drawing qualitative conclusions about the system from measurements, were reluctant to approximate the derivative through measurement. This pilot study led us to further questions. How do fields differ in their experts' concept image of partial derivatives? What representations of partial derivatives are preferred by experts? We plan to address these questions by means of further interviews with a wider range of disciplinary experts.

This article focuses on a method that engineers have used to make designs failsafe. Safety has always been implicit in design, but without any scientific data to lead the way in countering terrorism, engineers will have to figure out who decides what the terrorists are going to do, and what loads are needed. Gaining an understanding of terrorist organizations combined with some standard risk factors will help engineers determine where counterterrorism applications must be made. Staying ahead of terrorism, on an engineering level, will require engineers to perform continual risk assessment as technology advances, and new applications for existing technology are practiced.

This article discusses about Nightline, a robotics competition in which teams of students and engineers built robots that competed against each other in a sports-style event known as FIRST (For Inspiration and Recognition of Science and Technology). Creativity, communication, project management, partnerships, and a fundamental re-energizing of the engineering spirit are among the benefits that engineers and their supervisors describe as side effects of their involvement. While access to a future employee pool may be the reason that many organizations decide to get involved with FIRST, those side benefits are the reason most of them continue to participate. In many respects, FIRST has been turning out to be a fountain of youth. When hiring engineers, defense contractors have the added hurdle of getting their employees security clearance.

The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 μg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.

Pharmaceuticals and personal care products (PPCPs) are discharged in municipal wastewater. Effects in aquatic organisms exposed to individual pharmaceuticals in the laboratory have raised concerns regarding the environmental impacts of PPCPs, yet environmental exposures are always to complex mixtures. In this study, adult zebrafish (Danio rerio) showed significantly decreased embryo production after a 6 week exposure to a pharmaceutical mixture (MIX; 0.5 and 10μgL(-1)) of acetaminophen, carbamazepine, gemfibrozil and venlafaxine and to diluted wastewater effluent (WWE; 5% and 25%). Atretic oocytes and altered ovarian histology were significantly increased in female zebrafish exposed to both concentrations of MIX or WWE, which indicates a direct effect on oocyte development that may account for reduced embryo production. Apoptosis within the thecal and granulosa cell layers was identified in female zebrafish with atresia. Exposures to MIX or WWE at both concentrations severely altered kidney proximal tubule morphology, but no histological impacts on other organs were observed. Exposure of embryos to MIX or WWE at the high concentration significantly increased the incidence of developmental abnormalities. Embryo mortality was elevated with exposure to the high concentration of MIX. These studies indicate that chronic exposure of fish to pharmaceutical mixtures and wastewater impacts reproduction and induces histopathological changes, similar to what we have previously seen with single compound exposures. These data suggest that fish populations exposed to pharmaceuticals discharged in wastewater are at risk of negative impacts to reproductive capacity and health.

Cytochrome P450s (CYPs) are important xenobiotic metabolizing proteins. While their functions are well understood in mammals, CYP function in non-mammalian vertebrate systems is much less defined, with function often inferred from mammalian data, assuming similar function across vertebrate species. In this study, we investigate whether in vivo treatment with known mammalian CYP inducers can alter the in vitro catalytic activity of fish microsomes using eleven fluorescent CYP-mediated substrates. We investigate the basal metabolism and induction potential for hepatic CYPs in two fish species, rainbow trout (Oncorhynchus mykiss) and killifish (Fundulus heteroclitus). Species differences were found in the baseline metabolism of these substrates. Killifish have significantly higher metabolic rates for all tested substrates except 7-benzyloxyquinoline and 7-benzyloxy-4-trifluoromethylcoumarin (both mammalian CYP3A substrates); significant differences were also seen between male and female killifish. Treatment with dexamethasone, pregnenolone-16alpha-carbonitrile, and rifampicin did not cause broad, measurable CYP induction in either fish species. In trout, dexamethasone (100 mg kg(-1)) significantly induced 3-cyano-7-ethoxycoumarin metabolism and rifampicin (100 mg kg(-1)) induced the dealkylation of 7-methoxyresorufin, although both were highly variable. Female killifish exposed to pregnenolone-16alpha-carbonitrile (100 mg kg(-1)) showed significantly higher metabolism of 7-pentoxyresorufin. Overall, dexamethasone, pregnenolone-16alpha-carbonitrile and rifampicin did not appear to consistently increase CYP activity in fish. Trout treated with 10 or 50 mg kg(-1) beta-naphthoflavone (BNF), a CYP1A inducer, showed significantly induced activity across almost all substrates tested, exceptions being 7-benzyloxyquinoline, 7-benzyloxy-4-trifluoromethylcoumarin and dibenzylfluorescein. 7-Methoxy-4-(aminomethyl)coumarin, a typical CYP2D substrate in mammals, was not metabolized by untreated fish liver microsomes; however, treatment with BNF significantly induced the metabolism of this substrate in trout. Induced substrate metabolism in BNF-treated microsomes was only correlated across selective substrates, suggesting that BNF induces multiple CYPs in fish liver. These include the known BNF inducible CYP1s plus a number of as yet unidentified fish CYPs. Overall, many of these catalytic assays could be valuable tools for identification of the function of specific CYP subfamilies and individual isoforms in fish.

Pharmaceuticals discharged in municipal wastewater are of emerging concern because of their potential for inducing biological effects in aquatic organisms. Selective serotonin reuptake inhibitors (SSRIs), pharmaceuticals prescribed to treat chronic depression, have been detected in receiving and wastewaters. Fluoxetine is a highly prescribed model SSRI used to assess impacts of antidepressants on aquatic organisms. In this study, in vitro hepatic fluoxetine metabolism was determined in several model fish species: rainbow trout, goldfish, zebrafish and killifish. Incubation of fluoxetine with hepatic microsomes from trout pre-treated with carbamazepine showed a time-dependant loss of fluoxetine, concomitant with an increase in norfluoxetine, the major mammalian demethylated metabolite. However, fluoxetine was not well metabolized in reactions with hepatic microsomes from untreated fish. Fluoxetine loss was greater than norfluoxetine production, indicating that norfluoxetine is not the predominant fluoxetine biotransformation product in fish. Furthermore, norfluoxetine was often undetected, possibly indicating that fluoxetine demethylation is a minor metabolic pathway in fish. Inter-species differences in fluoxetine metabolism were not evident because of high intra-species variability, although killifish appeared to have the highest hepatic metabolic capacity for fluoxetine. Fluoxetine metabolism in mammals is catalyzed by cytochrome P450 (CYP) enzymes. Trout were exposed to known CYP inducers, carbamazepine and 3-methylcholanthrene, to assess potential induction of hepatic fluoxetine metabolism. Microsomes from 3-methylcholanthrene treated fish did not induce detectable changes in fluoxetine concentrations in vitro, indicating that fish CYP1s are not involved in fluoxetine metabolism; the CYPs involved are still unclear. Identification of metabolites other than norfluoxetine warrants further investigation.

This article discusses the importance of Professional Engineering (PE) license in the United States. The debate in United States was sparked by a proposal last year to create a single US license for each of the engineering disciplines. The proposal was made to enhance the ability of engineers to transport their expertise across state lines as well as across national borders to better compete in the global marketplace. The high number of industry exemptions also contributes to the decline of licensed engineers. Although those exemptions are considerable for most engineering disciplines, in Texas and New York, for example, the number of exemptions is highest among electrical and mechanical engineers. Between 1993 and 1998, the number of engineers taking the Fundamentals of Engineering exam, a precursor usually required to apply for a PE license, shrank significantly. While most companies include money to support the professional growth of their employees in their annual budgets, licensing appears to be held in a separate, unrelated category.