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1. A pharmacology practical class for preclinical medical students was designed as a placebo-controlled, double-blind trial of two bronchodilator drugs. 2. Fenoterol hydrobromide (800 micrograms), ipratropium bromide (80 micrograms) and placebo (propellant only) were given by metered dose inhaler to 79 non-asthmatic volunteers. Their effects on FEV1, heart rate and tremor (assessed by the time taken to thread five sewing needles) were compared. 3. Both drugs caused a significant increase in FEV1: the largest group mean increase was 77 ml, recorded 15 min after fenoterol, and 103 ml, recorded 60 min after ipratropium. 4. Fenoterol also caused a mean increase of 8.7 beats min- 1 in heart rate, 5 min after inhalation. This effect was still apparent after 60 min. 5. Fenoterol appeared to prolong needle threading time in some individuals. 6. In subjects who inhaled fenoterol, there were no correlations between the increase in FEV1, the increase in heart rate, or the development of tremor. 7. It is concluded that inhaled fenoterol and ipratropium both cause bronchodilation in normal subjects. Systemic absorption of fenoterol is indicated by the rapid increase in heart rate. The bronchodilator effect of ipratropium suggests that resting airway calibre is governed partly by parasympathetic tone in normal subjects. 8. The bronchodilator and systemic effects of these drugs can be used to demonstrate pharmacological, therapeutic and statistical principles to medical students. [ABSTRACT FROM AUTHOR]

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds., (© 2024. The Author(s).)

Groundwater resources are vital to ecosystems and livelihoods. Excessive groundwater withdrawals can cause groundwater levels to decline 1-10 , resulting in seawater intrusion 11 , land subsidence 12,13 , streamflow depletion 14-16 and wells running dry 17 . However, the global pace and prevalence of local groundwater declines are poorly constrained, because in situ groundwater levels have not been synthesized at the global scale. Here we analyse in situ groundwater-level trends for 170,000 monitoring wells and 1,693 aquifer systems in countries that encompass approximately 75% of global groundwater withdrawals 18 . We show that rapid groundwater-level declines (>0.5 m year -1 ) are widespread in the twenty-first century, especially in dry regions with extensive croplands. Critically, we also show that groundwater-level declines have accelerated over the past four decades in 30% of the world's regional aquifers. This widespread acceleration in groundwater-level deepening highlights an urgent need for more effective measures to address groundwater depletion. Our analysis also reveals specific cases in which depletion trends have reversed following policy changes, managed aquifer recharge and surface-water diversions, demonstrating the potential for depleted aquifer systems to recover., (© 2024. The Author(s).)

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon ( Salmo salar ) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed "switched-diet". Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 10 7 cells mL -1 ) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C 18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism ( ch25ha and lipe ), pathogen recognition ( clec12b and tlr5 ), and immune effectors ( znrf1 and cish ) . In contrast, these transcripts did not significantly respond to the bacterin in the "switched-diet" group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response ( tnfrsf6b ) and dendritic cell maturation ( ccl13 ) were upregulated, and a chemokine receptor ( cxcr1 ) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3 , was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member ( clec3a ) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds , 5loxa , 5loxb , tlr5 , and cxcr1 ). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation., Competing Interests: RT and RB are former and current employees of Cargill Inc., respectively, but did not participate in the qPCR study design, fatty acid analysis, the result interpretation, and the decision to submit the manuscript for publication. NU participated in this study as a postdoctoral fellow at Memorial University of Newfoundland, but he is currently employed by Aquabounty Canada Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Emam, Eslamloo, Caballero-Solares, Lorenz, Xue, Umasuthan, Gnanagobal, Santander, Taylor, Balder, Parrish and Rise.)

Gill damage represents a significant challenge in the teleost fish aquaculture industry globally, due to the gill's involvement in several vital functions and direct contact with the surrounding environment. To examine the local and systemic effects accompanying gill damage (which is likely to negatively affect gill function) of Atlantic salmon, we performed a field sampling to collect gill and liver tissue after several environmental insults (e.g., harmful algal blooms). Before sampling, gills were visually inspected and gill damage was scored; gill scores were assigned from pristine [gill score 0 (GS0)] to severely damaged gills (GS3). Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged (i.e., GS2) gill tissue. Rank Products analysis (5% percentage of false-positives) identified 254 and 34 upregulated and downregulated probes, respectively, in GS2 compared with GS0. Differentially expressed probes represented genes associated with functions including gill remodeling, wound healing, and stress and immune responses. We performed gill and liver qPCR for all four gill damage scores using microarray-identified and other damage-associated biomarker genes. Transcripts related to wound healing (e.g., neb and klhl41b ) were significantly upregulated in GS2 compared with GS0 in the gills. Also, transcripts associated with immune and stress-relevant pathways were dysregulated (e.g., downregulation of snaclec 1-like and upregulation of igkv3 ) in GS2 compared with GS0 gills. The livers of salmon with moderate gill damage (i.e., GS2) showed significant upregulation of transcripts related to wound healing (i.e., chtop ), apoptosis (e.g., bnip3l ), blood coagulation (e.g., f2 and serpind1b ), transcription regulation (i.e., pparg ), and stress-responses (e.g., cyp3a27 ) compared with livers of GS0 fish. We performed principal component analysis (PCA) using transcript levels for gill and liver separately. The gill PCA showed that PC1 significantly separated GS2 from all other gill scores. The genes contributing most to this separation were pgam2 , des , neb , tnnt2 , and myom1. The liver PCA showed that PC1 significantly separated GS2 from GS0; levels of hsp70 , cyp3a27 , pparg , chtop , and serpind1b were the highest contributors to this separation. Also, hepatic acute phase biomarkers (e.g., serpind1b and f2 ) were positively correlated to each other and to gill damage. Gill damage-responsive biomarker genes and associated qPCR assays arising from this study will be valuable in future research aimed at developing therapeutic diets to improve farmed salmon welfare., Competing Interests: Authors RB, and RT, in the representation of Cargill, Incorporated, and BM, in representation of Cermaq, participated in the design of the trial but had no role in the design of the gene expression experiment, the data collection and analysis, the preparation of this manuscript, and the decision to submit the manuscript for publication. RT and BM are currently not working in Cargill, Incorporated and Cermaq, respectively. Also, NU is currently working at AquaBounty Canada, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Emam, Caballero-Solares, Xue, Umasuthan, Milligan, Taylor, Balder and Rise.)

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. In the present study, pre-adult L. salmonis -infected and non-infected salmon were intraperitoneally injected with either formalin-killed Aeromonas salmonicida bacterin (ASAL) or phosphate-buffered saline (PBS). Dorsal skin samples from each injection/infection group (PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice) were collected at 24 h post-injection and used for transcriptome profiling using a 44K salmonid microarray platform. Microarray results showed no clear inflammation gene expression signatures and revealed extensive gene repression effects by pre-adult lice (2,189 down and 345 up-regulated probes) in the PBS-injected salmon (PBS/lice vs . PBS/no lice), which involved basic cellular (e.g., RNA and protein metabolism) processes. Lice repressive effects were not observed within the group of ASAL-injected salmon (ASAL/lice vs . ASAL/no lice); on the contrary, the observed skin transcriptome changes -albeit of lesser magnitude (82 up and 1 down-regulated probes)- suggested the activation in key immune and wound healing processes (e.g., neutrophil degranulation, keratinocyte differentiation). The molecular skin response to ASAL was more intense in the lice-infected (ASAL/lice vs . PBS/lice; 272 up and 11 down-regulated probes) than in the non-infected fish (ASAL/no lice vs . PBS/no lice; 27 up-regulated probes). Regardless of lice infection, the skin's response to ASAL was characterized by the putative activation of both antibacterial and wound healing pathways. The transcriptomic changes prompted by ASAL+lice co-stimulation (ASAL/lice vs . PBS/no lice; 1878 up and 3120 down-regulated probes) confirmed partial mitigation of lice repressive effects on fundamental cellular processes and the activation of pathways involved in innate (e.g., neutrophil degranulation) and adaptive immunity (e.g., antibody formation), as well as endothelial cell migration. The qPCR analyses evidenced immune-relevant genes co-stimulated by ASAL and lice in an additive (e.g., mbl2b , bcl6 ) and synergistic (e.g., hampa , il4r ) manner. These results provided insight on the physiological response of the skin of L. salmonis -infected salmon 24 h after ASAL stimulation, which revealed immunostimulatory properties by the bacterin with potential applications in anti-lice treatments for aquaculture. As a simulated co-infection model, the present study also serves as a source of candidate gene biomarkers for sea lice and bacterial co-infection., Competing Interests: RT and SS are former and current employees of Cargill Inc., respectively, but did not participate in the microarray/qPCR study design, the result interpretation, and the decision to submit the manuscript for publication. NU participated in this study as a postdoctoral fellow at Memorial University but was recently employed by Aquabounty Canada Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Caballero-Solares, Umasuthan, Xue, Katan, Kumar, Westcott, Chen, Fast, Skugor, Taylor and Rise.)

Sea lice ( Lepeophtheirus salmonis ) are ectoparasitic copepods that cause significant economic loss in marine salmoniculture. In commercial salmon farms, infestation with sea lice can enhance susceptibility to other significant pathogens, such as the highly contagious infectious salmon anemia virus (ISAv). In this study, transcriptomic analysis was used to evaluate the impact of four experimental functional feeds (i.e. 0.3% EPA/DHA+high-ω6, 0.3% EPA/DHA+high-ω6+immunostimulant (IS), 1% EPA/DHA+high-ω6, and 1% EPA/DHA+high-ω3) on Atlantic salmon ( Salmo salar ) during a single infection with sea lice ( L. salmonis ) and a co-infection with sea lice and ISAv. The overall objectives were to compare the transcriptomic profiles of skin between lice infection alone with co-infection groups and assess differences in gene expression response among animals with different experimental diets. Atlantic salmon smolts were challenged with L. salmonis following a 28-day feeding trial. Fish were then challenged with ISAv at 18 days post-sea lice infection (dpi), and maintained on individual diets, to establish a co-infection model. Skin tissues sampled at 33 dpi were subjected to RNA-seq analysis. The co-infection's overall survival rates were between 37%-50%, while no mortality was observed in the single infection with lice. With regard to the infection status, 756 and 1303 consensus differentially expressed genes (DEGs) among the four diets were identified in "lice infection vs. pre-infection" and "co-infection vs. pre-infection" groups, respectively, that were shared between the four experimental diets. The co-infection groups (co-infection vs. pre-infection) included up-regulated genes associated with glycolysis, the interferon pathway, complement cascade activity, and heat shock protein family, while the down-regulated genes were related to antigen presentation and processing, T-cell activation, collagen formation, and extracellular matrix. Pathway enrichment analysis conducted between infected groups (lice infection vs. co-infection) resulted in several immune-related significant GO terms and pathways unique to this group, such as "autophagosome", "cytosolic DNA-sensing pathway" and "response to type I interferons". Understanding how experimental functional feeds can impact the host response and the trajectory of co-infections will be an essential step in identifying efficacious intervention strategies that account for the complexities of disease in open cage culture., Competing Interests: Authors RGT and RB were employed by the company Cargill Innovation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2022 Cai, Kumar, Navaneethaiyer, Caballero-Solares, Carvalho, Whyte, Purcell, Gagne, Hori, Allen, Taylor, Balder, Parrish, Rise and Fast.)

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis , is one of the most devastating diseases of salmonids. However, the transcriptomic responses of Atlantic salmon ( Salmon salar ) in freshwater to an EM-90-like isolate have not been explored. Here, we infected Atlantic salmon parr with an EM-90-like isolate and conducted time-course qPCR analyses of pathogen load and four biomarkers ( campb , hampa , il8a , tlr5a ) of innate immunity on the head kidney samples. Transcript expression of three of these genes (except hampa ), as well as pathogen level, peaked at 21 days post-injection (DPI). Multivariate analyses of infected individuals at 21 DPI revealed two infection phenotypes [lower (L-SRS) and higher (H-SRS) infection level]. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling using a 44K salmonid microarray platform. We identified 1,636 and 3,076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups compared with the control group, respectively (FDR = 1%). Gene ontology term enrichment analyses of SRS-responsive genes revealed the activation of a large number of innate (e.g. "phagocytosis", "defense response to bacterium", "inflammatory response") and adaptive (e.g. "regulation of T cell activation", "antigen processing and presentation of exogenous antigen") immune processes, while a small number of general physiological processes (e.g. "apoptotic process", development and metabolism relevant) was enriched. Transcriptome results were confirmed by qPCR analyses of 42 microarray-identified transcripts. Furthermore, the comparison of individuals with differing levels of infection (H-SRS vs. L-SRS) generated insights into the biological processes possibly involved in disease resistance or susceptibility. This study demonstrated a low mortality (~30%) EM-90-like infection model and broadened the current understanding of molecular pathways underlying P. salmonis -triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen., Competing Interests: Authors EJ, SS, CH and RGT were employed by company Cargill Incorporated. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from EWOS Innovation (now part of Cargill, Incorporated). Authors EJ, SS, CH, and RGT, in the representation of Cargill, Incorporated, participated in the design of infection trial, sample collection, data interpretation, and manuscript preparation. However, they had no role in the design, data collection and analysis of gene expression experiments, and the decision to submit the manuscript for publication., (Copyright © 2021 Xue, Caballero-Solares, Hall, Umasuthan, Kumar, Jakob, Skugor, Hawes, Santander, Taylor and Rise.)

The importance of dietary omega-6 to omega-3 (ω6:ω3) fatty acid (FA) ratios for human health has been extensively examined. However, its impact on fish physiology, and the underlying molecular mechanisms, are less well understood. This study investigated the influence of plant-based diets (12-week exposure) with varying ω6:ω3 (0.4-2.7) on the hepatic transcriptome of Atlantic salmon. Using 44 K microarray analysis, genes involved in immune and inflammatory response ( lect2a , itgb5 , helz2a , p43 ), lipid metabolism ( helz2a ), cell proliferation ( htra1b ), control of muscle and neuronal development ( mef2d ) and translation ( eif2a , eif4b1 , p43 ) were identified; these were differentially expressed between the two extreme ω6:ω3 dietary treatments (high ω6 vs. high ω3) at week 12. Eight out of 10 microarray-identified transcripts showed an agreement in the direction of expression fold-change between the microarray and qPCR studies. The PPARα activation-related transcript helz2a was confirmed by qPCR to be down-regulated by high ω6 diet compared with high ω3 diet. The transcript expression of two helz2 paralogues was positively correlated with ω3, and negatively with ω6 FA in both liver and muscle, thus indicating their potential as biomarkers of tissue ω6:ω3 variation. Mef2d expression in liver was suppressed in the high ω6 compared to the balanced diet (ω6:ω3 of 2.7 and 0.9, respectively) fed fish, and showed negative correlations with ω6:ω3 in both tissues. The hepatic expression of two lect2 paralogues was negatively correlated with viscerosomatic index, while htra1b correlated negatively with salmon weight gain and condition factor. Finally, p43 and eif2a were positively correlated with liver Σω3, while these transcripts and eif4b2 showed negative correlations with 18:2ω6 in the liver. This suggested that some aspects of protein synthesis were influenced by dietary ω6:ω3. In summary, this nutrigenomic study identified hepatic transcripts responsive to dietary variation in ω6:ω3, and relationships of transcript expression with tissue (liver, muscle) lipid composition and other phenotypic traits.

The interaction of dietary eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA) levels with omega-6 to omega-3 ratios (ω6:ω3), and their impact on head kidney lipid metabolism in farmed fish, are not fully elucidated. We investigated the influence of five plant-based diets (12-week exposure) with varying EPA+DHA levels (0.3, 1.0, or 1.4%) and ω6:ω3 (high ω6, high ω3, or balanced) on tissue lipid composition, and transcript expression of genes involved in fatty acid and eicosanoid metabolism in Atlantic salmon head kidney. Tissue fatty acid composition was reflective of the diet with respect to C 18 PUFA and MUFA levels (% of total FA), and ω6:ω3 (0.5-1.5). Fish fed 0.3% EPA+DHA with high ω6 (0.3% EPA+DHA↑ω6) had the highest increase in proportions (1.7-2.3-fold) and in concentrations (1.4-1.8-fold) of arachidonic acid (ARA). EPA showed the greatest decrease in proportion and in concentration (by ~½) in the 0.3% EPA+DHA↑ω6 fed fish compared to the other treatments. However, no differences were observed in EPA proportions among salmon fed the high ω3 (0.3 and 1.0% EPA+DHA) and balanced (1.4% EPA+DHA) diets, and DHA proportions were similar among all treatments. Further, the transcript expression of elovl5a was lowest in the 0.3% EPA+DHA↑ω6 fed fish, and correlated positively with 20:3ω3, 20:4ω3 and EPA:ARA in the head kidney. This indicates that high dietary 18:3ω3 promoted the synthesis of ω3 LC-PUFA. Dietary EPA+DHA levels had a positive impact on elovl5a, fadsd5 and srebp1 expression, and these transcripts positively correlated with tissue ΣMUFA. This supported the hypothesis that LC-PUFA synthesis is positively influenced by tissue MUFA levels in Atlantic salmon. The expression of pparaa was higher in the 0.3% EPA+DHA↑ω6 compared to the 0.3% EPA+DHA↑ω3 fed fish. Finally, significant correlations between head kidney fatty acid composition and the expression of eicosanoid synthesis-related transcripts (i.e., 5loxa, 5loxb, cox1, cox2, ptges2, ptges3 , and pgds ) illustrated the constitutive relationships among fatty acids and eicosanoid metabolism in salmon., Competing Interests: RT was employed by the company Cargill, but did not have any additional role in the study design, data collection, analysis and interpretation, writing of the manuscript, or in the decision to submit the manuscript for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Katan, Xue, Caballero-Solares, Taylor, Rise and Parrish.)

Fluorescent natural organic matter at tryptophan-like (TLF) and humic-like fluorescence (HLF) peaks is associated with the presence and enumeration of faecal indicator bacteria in groundwater. We hypothesise, however, that it is predominantly extracellular material that fluoresces at these wavelengths, not bacterial cells. We quantified total (unfiltered) and extracellular (filtered at < 0.22 µm) TLF and HLF in 140 groundwater sources across a range of urban population densities in Kenya, Malawi, Senegal, and Uganda. Where changes in fluorescence occurred following filtration they were correlated with potential controlling variables. A significant reduction in TLF following filtration (ΔTLF) was observed across the entire dataset, although the majority of the signal remained and thus considered extracellular (median 96.9%). ΔTLF was only significant in more urbanised study areas where TLF was greatest. Beneath Dakar, Senegal, ΔTLF was significantly correlated to total bacterial cells (ρ s  0.51). No significant change in HLF following filtration across all data indicates these fluorophores are extracellular. Our results suggest that TLF and HLF are more mobile than faecal indicator bacteria and larger pathogens in groundwater, as the predominantly extracellular fluorophores are less prone to straining. Consequently, TLF/HLF are more precautionary indicators of microbial risks than faecal indicator bacteria in groundwater-derived drinking water.

Atlantic salmon smolts (approx. 20-months old) were fed experimental diets with different combinations of omega-6:omega-3 fatty acids (FAs) (high-ω6, high-ω3, or balanced) and eicosapentaenoic acid plus docosahexaenoic acid (EPA + DHA) levels (0.3, 1.0 or 1.4%) for 12 weeks. Muscle FA (% total FA) reflected dietary C 18 -polyunsaturated FA; however, muscle EPA per cent and content (mg g -1 ) were not different in salmon fed high-ω3 or balanced diets. Muscle DHA per cent was similar among treatments, while DHA content increased in fish fed 1.4% EPA + DHA, compared with those fed 0.3-1.0% EPA + DHA combined with high-ω6 FA. Muscle 20:3 ω 6 (DGLA) content was highest in those fed high-ω6 with 0.3% EPA + DHA. Quantitative polymerase chain reaction analyses on liver RNA showed that the monounsaturated FA synthesis-related gene, scdb, was upregulated in fish fed 1.0% EPA + DHA with high-ω6 compared to those fed 0.3% EPA + DHA. In high-ω3-fed salmon, liver elovl2 transcript levels were higher with 0.3% EPA + DHA than with 1.0% EPA + DHA. In high-ω6-fed fish, elovl2 did not vary with EPA + DHA levels, but it was positively correlated with muscle ARA, 22:4 ω 3 and DGLA. These results suggest dietary 18:3 ω 3 elongation contributed to maintaining muscle EPA + DHA levels despite a two- to threefold change in dietary proportions, while 18:2 ω 6 with 0.3% EPA + DHA increased muscle DGLA more than arachidonic acid (ARA). Positive correlations between hepatic elovl2 and fabp10a with muscle ω 6: ω 3 and EPA + DHA + ARA, respectively, were confirmed by reanalysing data from a previous salmon trial with lower variations in dietary EPA + DHA and ω 6: ω 3 ratios. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Parasitic sea lice (e.g., Lepeophtheirus salmonis ) cause costly outbreaks in salmon farming. Molecular insights into parasite-induced host responses will provide the basis for improved management strategies. We investigated the early transcriptomic responses in pelvic fins of Atlantic salmon parasitized with chalimus I stage sea lice. Fin samples collected from non-infected (i.e. pre-infected) control (PRE) and at chalimus-attachment sites (ATT) and adjacent to chalimus-attachment sites (ADJ) from infected fish were used in profiling global gene expression using 44 K microarrays. We identified 6568 differentially expressed probes (DEPs, FDR < 5%) that included 1928 shared DEPs between ATT and ADJ compared to PRE. The ATT versus ADJ comparison revealed 90 DEPs, all of which were upregulated in ATT samples. Gene ontology/pathway term network analyses revealed profound changes in physiological processes, including extracellular matrix (ECM) degradation, tissue repair/remodeling and wound healing, immunity and defense, chemotaxis and signaling, antiviral response, and redox homeostasis in infected fins. The QPCR analysis of 37 microarray-identified transcripts representing these functional themes served to confirm the microarray results with a significant positive correlation ( p < 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon's fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon's attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for L. salmonis ., Competing Interests: Authors S.S. and R.G.T. in the representation of Cargill Innovation, participated in the data interpretation and discussion, but had no role in the design of the microarray experiment and transcript expression experiment, the data collection and analysis, the preparation of this manuscript, and the decision to submit the manuscript for publication. All authors declare no conflict of interest.

MicroRNAs (miRNAs) are key regulators in fish immune responses. However, no study has previously characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida (ASAL) on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. To this end, first, we performed small RNA deep sequencing and qPCR analyses to identify and confirm pIC- and/or ASAL-responsive miRNAs in the head kidney of salmon fed a control diet. DESeq2 analyses identified 12 and 18 miRNAs differentially expressed in pIC and ASAL groups, respectively, compared to the controls. Fifteen of these miRNAs were studied by qPCR; nine remained significant by qPCR. Five miRNAs (miR-27d-1-2-5p, miR-29b-2-5p, miR-146a-5p, miR-146a-1-2-3p, miR-221-5p) were shown by qPCR to be significantly induced by both pIC and ASAL. Second, the effect of CpG-containing functional feed on miRNA expression was investigated by qPCR. In pre-injection samples, 6 of 15 miRNAs (e.g., miR-181a-5-3p, miR-462a-3p, miR-722-3p) had significantly lower expression in fish fed CpG diet than control diet. In contrast, several miRNAs (e.g., miR-146a-1-2-3p, miR-192a-5p, miR-194a-5p) in the PBS- and ASAL-injected groups had significantly higher expression in CpG-fed fish. Multivariate statistical analyses confirmed that the CpG diet had a greater impact on miRNA expression in ASAL-injected compared with pIC-injected fish. This study identified immune-relevant miRNA biomarkers that will be valuable in the development of diets to combat infectious diseases of salmon.

Groundwater in sub-Saharan Africa supports livelihoods and poverty alleviation 1,2 , maintains vital ecosystems, and strongly influences terrestrial water and energy budgets 3 . Yet the hydrological processes that govern groundwater recharge and sustainability-and their sensitivity to climatic variability-are poorly constrained 4,5 . Given the absence of firm observational constraints, it remains to be seen whether model-based projections of decreased water resources in dry parts of the region 4 are justified. Here we show, through analysis of multidecadal groundwater hydrographs across sub-Saharan Africa, that levels of aridity dictate the predominant recharge processes, whereas local hydrogeology influences the type and sensitivity of precipitation-recharge relationships. Recharge in some humid locations varies by as little as five per cent (by coefficient of variation) across a wide range of annual precipitation values. Other regions, by contrast, show roughly linear precipitation-recharge relationships, with precipitation thresholds (of roughly ten millimetres or less per day) governing the initiation of recharge. These thresholds tend to rise as aridity increases, and recharge in drylands is more episodic and increasingly dominated by focused recharge through losses from ephemeral overland flows. Extreme annual recharge is commonly associated with intense rainfall and flooding events, themselves often driven by large-scale climate controls. Intense precipitation, even during years of lower overall precipitation, produces some of the largest years of recharge in some dry subtropical locations. Our results therefore challenge the 'high certainty' consensus regarding decreasing water resources 4 in such regions of sub-Saharan Africa. The potential resilience of groundwater to climate variability in many areas that is revealed by these precipitation-recharge relationships is essential for informing reliable predictions of climate-change impacts and adaptation strategies.

Competing Interests: Competing interests: None declared.

The transforming growth factor β (TGF β ) superfamily includes TGF β , activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGF β superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor β receptor 1 (TGFBR1) kinase inhibitors designed to target TGF β signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC 50 = 18 and 47 nM, respectively) were more effective inhibitors of TGF β -induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC 50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC 50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGF β -induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGF β - and BMP-regulated signaling pathways to disease states., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Dependence on marine natural resources threatens the sustainability of Atlantic salmon aquaculture. In the present study, Atlantic salmon fed for 14 weeks with an experimental diet based on animal by-products and vegetable oil (ABP) exhibited reduced growth performance compared with others fed a fish meal/fish oil based experimental diet (MAR) and a plant protein/vegetable oil-based experimental diet (VEG). To characterize the molecular changes underlying the differences in growth performance, we conducted a 44 K microarray study of the liver transcriptome of the three dietary groups., Results: The microarray experiment identified 122 differentially expressed features (Rank Products, PFP < 10%). Based on their associated Gene Ontology terms, 46 probes were classified as metabolic and growth-relevant genes, 25 as immune-related, and 12 as related to oxidation-reduction processes. The microarray results were validated by qPCR analysis of 29 microarray-identified transcripts. Diets significantly modulated the transcription of genes involved in carbohydrate metabolism (gck and pfkfb4), cell growth and proliferation (sgk2 and htra1), apoptosis (gadd45b), lipid metabolism (fabp3, idi1, sqs), and immunity (igd, mx, ifit5, and mhcI). Hierarchical clustering and linear correlation analyses were performed to find gene expression patterns among the qPCR-analyzed transcripts, and connections between them and muscle and liver lipid composition. Overall, our results indicate that changes in the liver transcriptome and tissue lipid composition were driven by cholesterol synthesis up-regulation by ABP and VEG diets, and the lower carbohydrate intake in the ABP group. Two of the microarray-identified genes (sgk2 and htra1) might be key to explaining glucose metabolism regulation and the dietary-modulation of the immune system in fish. To evaluate the potential of these genes as predictive biomarkers, we subjected the qPCR data to a stepwise discriminant analysis. Three sets of no more than four genes were found to be able to predict, with high accuracy (67-94%), salmon growth and fatty acid composition., Conclusions: This study provides new findings on the impact of terrestrial animal and plant products on the nutrition and health of farmed Atlantic salmon, and a new method based on gene biomarkers for potentially predicting desired phenotypes, which could help formulate superior feeds for the Atlantic salmon aquaculture industry.

Due to limited fish meal and fish oil resources and their high costs for the aquaculture industry, it is necessary to find alternative sustainable sources of protein and lipids. Therefore, seven different diets were formulated with different levels of animal by-products, vegetable proteins, fish oil and rapeseed oil, to feed farmed Atlantic salmon, and their effects on growth performance, muscle lipid class, and fatty acid composition were examined. Protein sources included anchovy, poultry, feather, blood, corn, soy and wheat. Growth performance indicated that the diet with the lowest fish meal and fish oil content resulted in the lowest weight gain and final weight, followed by the diet containing the highest level of animal by-products. The lipid class analysis showed no statistical difference in the muscle total lipid content using different diets. However, significant statistical differences were observed among the main lipid classes; triacylglycerols, phospholipids, and sterols. The diet containing 1.4% omega-3 long-chain fatty acids resulted in the highest content of triacylglycerols and phospholipids. Diets containing medium and low levels of fish oil and fish meal, respectively, led to as high a level of ω3 fatty acids in muscle as when fish were fed diets with high levels of fish meal and fish oil. The results of this study suggest that feeding a diet containing low levels of fish meal and moderate levels of fish oil does not significantly affect ω3 fatty acid composition in muscle. Fish meal could be reduced to 5% without affecting growth as long as there was a minimum of 5% fish oil, and animal by-products did not exceed 26% of the diet., Competing Interests: Cargill provided support in the form of salary for author RT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Cutaneous squamous cell carcinoma (cSCC) has a high tumour mutational burden (50 mutations per megabase DNA pair). Here, we combine whole-exome analyses from 40 primary cSCC tumours, comprising 20 well-differentiated and 20 moderately/poorly differentiated tumours, with accompanying clinical data from a longitudinal study of immunosuppressed and immunocompetent patients and integrate this analysis with independent gene expression studies. We identify commonly mutated genes, copy number changes and altered pathways and processes. Comparisons with tumour differentiation status suggest events which may drive disease progression. Mutational signature analysis reveals the presence of a novel signature (signature 32), whose incidence correlates with chronic exposure to the immunosuppressive drug azathioprine. Characterisation of a panel of 15 cSCC tumour-derived cell lines reveals that they accurately reflect the mutational signatures and genomic alterations of primary tumours and provide a valuable resource for the validation of tumour drivers and therapeutic targets.

Background: Due to the limited availability and high cost of fish oil in the face of increasing aquaculture production, there is a need to reduce usage of fish oil in aquafeeds without compromising farm fish health. Therefore, the present study was conducted to determine if different levels of vegetable and fish oils can alter antiviral responses of salmon macrophage-like cells (MLCs). Atlantic salmon (Salmo salar) were fed diets containing 7.4% (FO7) or 5.1% (FO5) fish oil. These diets were designed to be relatively low in EPA + DHA (i.e. FO7: 1.41% and FO5: 1%), but near the requirement level, and resulting in comparable growth. Vegetable oil (i.e. rapeseed oil) was used to balance fish oil in experimental diets. After a 16-week feeding trial, MLCs isolated from fish in these dietary groups were stimulated by a viral mimic (dsRNA: pIC) for 6 h (qPCR assay) and 24 h (microarray and qPCR assays)., Results: The fatty acid composition of head kidney leukocytes varied between the two dietary groups (e.g. higher 20:5n-3 in the FO7 group). Following microarray assays using a 44K salmonid platform, Rank Products (RP) analysis showed 14 and 54 differentially expressed probes (DEP) (PFP < 0.05) between the two diets in control and pIC groups (FO5 vs. FO7), respectively. Nonetheless, Significance Analysis of Microarrays (SAM, FDR < 0.05) identified only one DEP between pIC groups of the two diets. Moreover, we identified a large number (i.e. 890 DEP in FO7 and 1128 DEP in FO5 overlapping between SAM and RP) of pIC-responsive transcripts, and several of them were involved in TLR-/RLR-dependent and cytokine-mediated pathways. The microarray results were validated as significantly differentially expressed by qPCR assays for 2 out of 9 diet-responsive transcripts and for all of the 35 selected pIC-responsive transcripts., Conclusion: Fatty acid-binding protein adipocyte (fabp4) and proteasome subunit beta type-8 (psmb8) were significantly up- and down-regulated, respectively, in the MLCs of fish fed the diet with a lower level of fish oil, suggesting that they are important diet-responsive, immune-related biomarkers for future studies. Although the different levels of dietary fish and vegetable oils involved in this study affected the expression of some transcripts, the immune-related pathways and functions activated by the antiviral response of salmon MLCs in both groups were comparable overall. Moreover, the qPCR revealed transcripts responding early to pIC (e.g. lgp2, map3k8, socs1, dusp5 and cflar) and time-responsive transcripts (e.g. scarb1-a, csf1r, traf5a, cd80 and ctsf) in salmon MLCs. The present study provides a comprehensive picture of the putative molecular pathways (e.g. RLR-, TLR-, MAPK- and IFN-associated pathways) activated by the antiviral response of salmon MLCs.

Groundwater-level fluctuations represent hydraulic responses to changes in groundwater storage due to aquifer recharge and drainage as well as to changes in stress that include water mass loading and unloading above the aquifer surface. The latter 'poroelastic' response of confined aquifers is a well-established phenomenon which has been demonstrated in diverse hydrogeological environments but is frequently ignored in assessments of groundwater resources. Here we present high-frequency groundwater measurements over a twelve-month period from the tropical, fluvio-deltaic Bengal Aquifer System (BAS), the largest aquifer in south Asia. The groundwater level fluctuations are dominated by the aquifer poroelastic response to changes in terrestrial water loading by processes acting over periods ranging from hours to months; the effects of groundwater flow are subordinate. Our measurements represent the first direct, quantitative identification of loading effects on groundwater levels in the BAS. Our analysis highlights the potential limitations of hydrogeological analyses which ignore loading effects in this environment. We also demonstrate the potential for employing poroelastic responses in the BAS and across other tropical fluvio-deltaic regions as a direct, in-situ measure of changes in terrestrial water storage to complement analyses from the Gravity and Climate Experiment (GRACE) mission but at much higher resolution.

Two novel tetrathiafulvalene (TTF) containing compounds 1 and 2 have been synthesised via a four-fold Stille coupling between a tetrabromo-dithienoTTF 5 and stannylated thiophene 6 or thiazole 4. The optical and electrochemical properties of compounds 1 and 2 have been measured by UV-vis spectroscopy and cyclic voltammetry and the results compared with density functional theory (DFT) calculations to confirm the observed properties. Organic field effect transistor (OFET) devices fabricated from 1 and 2 demonstrated that the substitution of thiophene units for thiazoles was found to increase the observed charge transport, which is attributed to induced planarity through S-N interactions of adjacent thiazole nitrogen atoms and TTF sulfur atoms and better packing in the bulk.

Objectives: Important barriers to addressing the sexually transmitted infection (STI) epidemic among adolescents are the inadequate partner notification of positive STI results and insufficient rates of partner testing and treatment. However, adolescent attitudes regarding partner notification and treatment are not well understood. The aim was to qualitatively explore the barriers to and preferences for partner notification and treatment among adolescent males and females tested for STIs in an emergency department (ED) setting and to explore the acceptability of ED personnel notifying their sexual partners., Methods: This was a descriptive, qualitative study in which a convenience sample of 40 adolescents (18 females, 22 males) 14 to 21 years of age who presented to either adult or pediatric EDs with STI-related complaints participated. Individualized, semistructured, confidential interviews were administered to each participant. Interviews were audiotaped and transcribed verbatim by an independent transcriptionist. Data were analyzed using framework analysis., Results: Barriers to partner notification included fear of retaliation or loss of the relationship, lack of understanding of or concern for the consequences associated with an STI, and social stigma and embarrassment. Participants reported two primary barriers to their partners obtaining STI testing and treatment: lack of transportation to the health care site and the partner's fear of STI positive test results. Most participants preferred to notify their main sexual partners of an STI exposure via a face-to-face interaction or a phone call. Most participants were agreeable with a health care provider (HCP) notifying their main sexual partners of STI exposure and preferred that the HCP notify the partner by phone call., Conclusions: There are several adolescent preferences and barriers for partner notification and treatment. To be most effective, future interventions to prevent adolescent STIs should incorporate these preferences and address the barriers to partner notification. In an ED setting, using HCPs to provide partner notification of STI exposures is acceptable to adolescent patients; however, the feasibility of this type of program needs further exploration., (© 2014 by the Society for Academic Emergency Medicine.)

We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 μM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The design of a new class of materials, called organic molecules of intrinsic microporosity (OMIMs), incorporates awkward, concave shapes to prevent efficient packing of molecules, resulting in microporosity. This work presents predictive molecular simulations and experimental wide-angle X-ray scattering (WAXS) for a series of biphenyl-core OMIMs with varying end-group geometries. Development of the utilized simulation protocol was based on comparison of several simulation methods to WAXS patterns. In addition, examination of the simulated structures has facilitated the assignment of WAXS features to specific intra- and intermolecular distances, making this a useful tool for characterizing the packing behavior of this class of materials. Analysis of the simulations suggested that OMIMs had greater microporosity when the molecules were the most shape-persistent, which required rigid structures and bulky end groups. The simulation protocol presented here allows for predictive, presynthesis screening of OMIMs and similar complex molecules to enhance understanding of their structures and aid in future design efforts.

Three travel cost models are used to estimate non-fishing recreation demand at the Snake River reservoirs recreation area in eastern Washington. The opportunity cost of time is specified in the "traditional" and McConnell-Strand models as a fraction of the exogenous market wage rate and in the two-step decision model as a function of socioeconomic attributes of the recreationists. Benefits per person per trip were $90, $35, and $46 respectively, for the three models. Boaters visit the site more than three times as often as non-boaters resulting in higher annual benefits for boaters., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Context: The extent to which 25-hydroxyvitamin D [25(OH)D] and IGF-I influence bone mineral content (BMC) accrual from early to mid-puberty is unclear. OBJECTIVE, SETTING, AND PARTICIPANTS: This study sought to determine relationships among 25(OH)D, IGF-I, and BMC in community-dwelling prepubertal females (n = 76; aged 4-8 yr at baseline) over a period of up to 9 yr., Design: The hypothesis that changes in IGF-I vs. 25(OH)D are more strongly associated with BMC accrual was formulated after data collection. 25(OH)D and IGF-I were log-transformed and further adjusted using two-way ANOVA for differences in season and race. Linear mixed modeling (including a random subject-specific intercept and a random subject-specific slope on age) was employed to analyze the proportion of variance the transformed 25(OH)D and IGF-I variables explained for the bone outcomes., Results: IGF-I was more strongly associated with BMC accrual than 25(OH)D at the total body (R(2) = 0.874 vs. 0.809), proximal femur (R(2) = 0.847 vs. 0.771), radius (R(2) = 0.812 vs. 0.759), and lumbar spine (R(2) = 0.759 vs. 0.698). The rate of BMC accrual was positively associated with changes in IGF-I but negatively associated with 25(OH)D. When IGF-I and 25(OH)D were included in the same regression equation, 25(OH)D did not have a significant predictive effect on BMC accrual above and beyond that of IGF-I., Conclusions: These prospective data in early adolescent females indicate that both 25(OH)D and IGF-I have a significant impact on bone mineral accrual; however, the positive association of IGF-I and BMC accrual is greater than the negative association of 25(OH)D and BMC accrual.

Background: Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR., Results: Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes., Conclusion: Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.

We have characterized and quantified a form of bacterial chemotaxis that manifests only as an emergent property by measuring symmetry breaking in a swarm of Myxococcus xanthus exposed to a two-dimensional nutrient gradient from within an agar substrate. M. xanthus chemotaxis requires cell-cell contact and coordinated motility, as individual motile cells exhibit only nonvectorial movement in the presence of a nutrient gradient. Genes that specifically affect M. xanthus chemotaxis include at least 10 of the 53 that express enhancer binding proteins of the NtrC-like class, an indication that this behavior is controlled through transcription, most likely by a complex signal transduction network.

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN&#95;5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN&#95;1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.

Accurate determination of functional interactions among proteins at the genome level remains a challenge for genomic research. Here we introduce a genome-scale approach to functional protein annotation--phylogenomic mapping--that requires only sequence data, can be applied equally well to both finished and unfinished genomes, and can be extended beyond single genomes to annotate multiple genomes simultaneously. We have developed and applied it to more than 200 sequenced bacterial genomes. Proteins with similar evolutionary histories were grouped together, placed on a three dimensional map and visualized as a topographical landscape. The resulting phylogenomic maps display thousands of proteins clustered in mountains on the basis of coinheritance, a strong indicator of shared function. In addition to systematic computational validation, we have experimentally confirmed the ability of phylogenomic maps to predict both mutant phenotype and gene function in the delta proteobacterium Myxococcus xanthus.

We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set. Analysis of the ncRNAs reveals a set that is highly conserved in chickens and mammals, including sequences for 14 pri-miRNAs encoding 23 different miRNAs. The data sets described here provide a transcriptome toolkit linked to physical clones for bioinformaticians and experimental biologists who wish to use chicken systems as a low-cost, accessible alternative to mammals for the analysis of vertebrate development, immunology, and cell biology.

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

Effects of stress on driving performance can depend on the nature of driver's stress reactions and on the traffic environment. In an experimental study, we assessed multiple dimensions of vulnerability to driver stress by a questionnaire that was validated in previous field studies and related those dimensions to performance on a driving simulator. Results were broadly consistent with prediction. A dimension of habitual dislike of driving was associated with reduced control skills, greater caution, and disturbance of moods. A measure of aggressive driving predicted more frequent and more error-prone overtaking, which are effects attributed to the use of confrontive coping strategies in interaction with other vehicles. An alertness measure predicted speed of reaction to pedestrian hazards. This research has practical applications for system design, automated monitoring of driver performance, selection and assessment of drivers, and training.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 microM TM or 0.1 microM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.

We have identified serine 254 as an essential residue in rat histidase (histidine ammonia-lyase, EC 4.3.1.3). Histidase and phenylalanine ammonia-lyase are the only two enzymes that have been postulated to require the modified amino acid, dehydroalanine, for enzyme activity. In the bacterial peptides nisin and subtilin, and in the pyruvoyl enzymes, the precursor for dehydroalanine is a serine. To determine whether serine may be the dehydroalanine precursor in rat histidase, we substituted four highly conserved serines with alanines, and expressed the mutated histidase cDNAs in COS cells, which have no endogenous histidase activity. Substitution of serines 223, 254, 508, and 533 resulted in the production of approximately equal amounts of histidase protein with histidase activities of 2.4, 0.0, 75, and 16%, respectively. The abrogation of histidase activity by the substitution of alanine for serine 254, together with the modification by L-cysteine of the corresponding residue in Pseudomonas putida histidase (Hernandez, C., Stroh, J. G., and Phillips, A. T. (1993) Arch. Biochem. Biophys. 307, 126-132), is strong evidence that this residue is the precursor of the essential electrophilic moiety of histidase.