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Gene transcript profiling in sea otters post-Exxon Valdez oil spill: A tool for marine ecosystem health assessment

Using a panel of genes stimulated by oil exposure in a laboratory study, we evaluated gene transcription in blood leukocytes sampled from sea otters captured from 2006–2012 in western Prince William Sound (WPWS), Alaska, 17–23 years after the 1989 Exxon Valdez oil spill (EVOS). We compared WPWS sea otters to reference populations (not affected by the EVOS) from the Alaska Peninsula (2009), Katmai National Park and Preserve (2009), Clam Lagoon at Adak Island (2012), Kodiak Island (2005) and captive sea otters in aquaria. Statistically, sea otter gene transcript profiles separated into three distinct clusters: Cluster 1, Kodiak and WPWS 2006–2008 (higher relative transcription); Cluster 2, Clam Lagoon and WPWS 2010–2012 (lower relative transcription); and Cluster 3, Alaska Peninsula, Katmai and captive sea otters (intermediate relative transcription). The lower transcription of the aryl hydrocarbon receptor (AHR), an established biomarker for hydrocarbon exposure, in WPWS 2010–2012 compared to earlier samples from WPWS is consistent with declining hydrocarbon exposure, but the pattern of overall low levels of transcription seen in WPWS 2010–2012 could be related to other factors, such as food limitation, pathogens or injury, and may indicate an inability to mount effective responses to stressors. Decreased transcriptional response across the entire gene panel precludes the evaluation of whether or not individual sea otters show signs of exposure to lingering oil. However, related studies on sea otter demographics indicate that by 2012, the sea otter population in WPWS had recovered, which indicates diminishing oil exposure.

Alaska

Gene expression profiles in two razor clam populations: Discerning drivers of population status

With rapidly changing marine ecosystems, shifts in abundance and distribution are being documented for a variety of intertidal species. We examined two adjacent populations of Pacific razor clams ( Siliqua patula ) in lower Cook Inlet, Alaska. One population (east) supported a sport and personal use fishery, but this has been closed since 2015 due to declines in abundance, and the second population (west) continues to support commercial and sport fisheries. We used gene expression to investigate potential causes of the east side decline, comparing razor clam physiological responses between east and west Cook Inlet. The target gene profile used was developed for razor clam populations in Alaska based on physiological responses to environmental stressors. In this study, we identified no differences of gene expression between east and west populations, leading to two potential conclusions: (1) differences in factors capable of influencing physiology exist between the east and west and are sufficient to influence razor clam populations but are not detected by the genes in our panel, or (2) physiological processes do not account for the differences in abundance, and other factors such as predation or changes in habitat may be impacting the east Cook Inlet population.

Alaska

Frozen gene pools - A future for species otherwise destined for extinction

Conclusion: Semen banks and ova and embryo banks can be practical methods to maintain gene pools. Gene pool preservation is desperately needed today due to the rapid decline in number of species and their habitat, a matter that is of concern to.biologists, economists, and politicians worldwide. Techniques are available for the cryopreservation of semen from many animals (and embryos from a few mammals) and adaptations of these techniques to other animals should be possible. A frozen gene pool in conjunction with existing programs makes it possible to preserve gene pools at less cost or in.some cases where no other alternative to extinction existed.

American Association of Zoological Parks and Aquar

Tools to minimize interlaboratory variability in vitellogenin gene expression monitoring programs

The egg yolk precursor protein vitellogenin is widely used as a biomarker of estrogen exposure in male fish. However, standardized methodology is lacking and little is known regarding the reproducibility of results among laboratories using different equipment, reagents, protocols, and data analysis programs. To address this data gap we tested the reproducibility across laboratories to evaluate vitellogenin gene ( vtg ) expression and assessed the value of using a freely available software data analysis program. Samples collected from studies of male fathead minnows ( Pimephales promelas ) exposed to 17α-ethinylestradiol (EE2) and minnows exposed to processed wastewater effluent were evaluated for vtg expression in 4 laboratories. Our results indicate reasonable consistency among laboratories if the free software for expression analysis LinRegPCR is used, with 3 of 4 laboratories detecting vtg in fish exposed to 5 ng/L EE2 ( n = 5). All 4 laboratories detected significantly increased vtg levels in 15 male fish exposed to wastewater effluent compared with 15 male fish held in a control stream. Finally, we were able to determine that the source of high interlaboratory variability from complementary deoxyribonucleic acid (cDNA) to quantitative polymerase chain reaction (qPCR) analyses was the expression analysis software unique to each real-time qPCR machine. We successfully eliminated the interlaboratory variability by reanalyzing raw fluorescence data with independent freeware, which yielded cycle thresholds and polymerase chain reaction (PCR) efficiencies that calculated results independently of proprietary software. Our results suggest that laboratories engaged in monitoring programs should validate their PCR protocols and analyze their gene expression data following the guidelines established in the present study for all gene expression biomarkers.

Environmental Toxicology and Chemistry

Microbial source tracking and land use associations for antibiotic resistance genes in private wells influenced by human and livestock fecal sources

Antimicrobial resistance is a growing public health problem that requires an integrated approach among human, agricultural, and environmental sectors. However, few studies address all three components simultaneously. We investigated the occurrence of five antibiotic resistance genes (ARGs) and the class 1 integron gene ( intI1 ) in private wells drawing water from a vulnerable aquifer influenced by residential septic systems and land-applied dairy manure. Samples ( n = 138) were collected across four seasons from a randomized sample of private wells in Kewaunee County, Wisconsin. Measurements of ARGs and intI1 were related to microbial source tracking (MST) markers specific to human and bovine feces; they were also related to 54 risk factors for contamination representing land use, rainfall, hydrogeology, and well construction. ARGs and intI1 occurred in 5–40% of samples depending on target. Detection frequencies for ARGs and intI1 were lowest in the absence of human and bovine MST markers (1-30%), highest when co-occurring with human and bovine markers together (11-78%), and intermediate when co-occurring with just one type of MST marker (4-46%). Gene targets were associated with septic system density more often than agricultural land, potentially because of the variable presence of manure on the landscape. Determining ARG prevalence in a rural setting with mixed land use allowed an assessment of the relative contribution of human and bovine fecal sources. Because fecal sources co-occurred with ARGs at similar rates, interventions intended to reduce ARG occurrence may be most effective if both sources are considered.

Wisconsin

Gene transcript profiling in desert bighorn sheep

Respiratory disease is a key factor affecting the conservation and recovery of bighorn sheep ( Ovis canadensis ) populations. Innovative, minimally invasive tools such as gene transcription–based diagnostics have the potential to improve our understanding of the broad range of factors that can affect the health of wild sheep. Evaluation of transcript levels for genes representative of multiple internal systems enables measurement of physiological responses of individuals as well as populations to environmental stressors such as pathogens, nutritional deficiency, or contaminants. We developed real‐time polymerase chain reaction assays for 14 genes of interest representing systems including inflammation, cell signaling, detoxification, antiviral, antibacterial, or general stress. Initial results from desert bighorn sheep ( O. c. nelsoni ) sampled from the River, Muddy, and Bare mountains as well as from the Pintwater Range, in southern Nevada, USA, indicated unique transcript profiles associated with each population. This initial study provides the framework from which controlled variable or longitudinal studies can be made, thus augmenting the potential to inform management actions in the future.

Nevada

Identification and regulatory analysis of rainbow trout tapasin and tapasin-related genes

Tapasin (TAPBP) is a key member of MHC class Ia antigen-loading complexes, bridging the class Ia molecule to the transporter associated with antigen presentation (TAP). As part of an ongoing study of MHC genomics in rainbow trout, we have identified two rainbow trout TAPBP genes (Onmy-TAPBP.a and .b) and a similar but distinct TAPBP-related gene (Onmy-TAPBP-R) that had previously only been described in mammals. Physical and genetic mapping indicate that Onmy-TAPBP.a is on chromosome 18 in the MHC class Ia region and that Onmy-TAPBP.b resides on chromosome 14 in the MHC class Ib region. There are also at least two copies of TAPBP-R, Onmy-TAPBP-R.a and Onmy-TAPBP-R.b, located on chromosomes 2 and 3, respectively. Due to the central role of TAPBP expression during acute viral infection, we have characterized the transcriptional profile and regulatory regions for both Onmy-TAPBP and Onmy-TAPBP-R. Transcription of both genes increased during acute infection with infectious hematapoeitic necrosis virus (IHNV) in a fashion indicative of interferon-mediated regulation. Promoter-reporter assays in STE-137 cells demonstrate that the trout TAPBP and TAPBP-R promoters respond to interferon regulatory factors, Onmy-IRF1 and Onmy-IRF2. Overall, TAPBP is expressed at higher levels than TAPBP-R in nai??ve tissues and TAPBP transcription is more responsive to viral infection and IRF1 and 2 binding. ?? Springer-Verlag 2006.

Immunogenetics

Genes in space: What Mojave desert tortoise genetics can tell us about landscape connectivity

Habitat loss and fragmentation in the Mojave Desert have been increasing, which can create barriers to movement and gene flow leading to decreased populations of native species. Disturbance and degradation of Mojave desert tortoise habitat includes linear features (e.g. highways, railways, and a network of dirt roads), urbanized areas, and their associated infrastructure, mining activities, energy distribution systems, and most recently, utility-scale solar facilities. To evaluate the spatial genetic structure of tortoises in an area experiencing rapid habitat loss, we conducted field surveys from 2015-2017 and genotyped 299 tortoises at 20 microsatellite loci within and around Ivanpah Valley along the California/Nevada border. We used a Bayesian clustering analysis to examine population genetic structure across valley and mountain pass habitat. Spatial principal components analysis was included to further investigate population genetic structure with isolation-by-distance. To explicitly incorporate landscape features (e.g. habitat and anthropogenic linear barriers) we used maximum likelihood population effects. We assessed recent gene flow on the landscape through maximum likelihood pedigree analyses of relatedness. We detected three to four genetic clusters with high levels of admixture that generally corresponded to three valleys separated by mountain ranges, and a genetically distinguishable population in one mountain pass. Pedigree analyses showed second order relationships up to 60 km apart suggesting a greater range of interactions and inter-relatedness between individuals than previously suspected. Our results support historical gene flow with isolation-by-resistance, and reveal a genetic signal indicative of reduction in genetic connectivity across two parallel linear features (a railway and a highway). This work demonstrates the value of protecting connected tracts of functional habitat and the importance of connectivity research in conservation.

California, Nevada

Genomic architecture and repertoire of the rainbow trout immunoglobulin light chain genes

The genomic loci encoding the four immunoglobulin light chains (IgL1, IgL2, IgL3, and IgL4) in the Swanson trout genome assembly were annotated in order to provide a measurement of the potential IgL repertoire. IgL1 and IgL3 gene segments are co-localized on chromosomes 21, 18, 15, and 7 while IgL2 and IgL4 were found on chromosomes 13 and 17, respectively. In total, 48 constant (C L ), 87 variable (V L ), and 59 joining (J L ) productive genes are described. Pairwise alignment of the V L segments revealed that they belong to nine different families, three of which (kappa IV, V, and VI) are described for the first time in this study. V L and C L sequences on chromosome 15 and 21 and those on chromosomes 7 and 18 clustered together in phylogenetic analysis. PCR was used to examine IgL C L and V L genes in 9 lines of rainbow trout. IgL4 in the Hot Creek and Golden trout lines was missing 42 nucleotides resulting in a loss of 14 amino acids. The sigma IV variable family was completely absent from the Swanson, Arlee, Hot Creek, and wild type lines and silenced in the Skamania line with the addition of 176 bp mini-satellite insert. Similarly, the Whale Rock, Arlee, and wild type lines were all found to encode two sigma II products, a functional 252 bp product and a larger 425 bp product that contained a 172 bp insert. Results from this study indicate that there are genomic differences in IgL repertoire between different lines of trout that could affect humoral immune responses post vaccination and during disease.

Developmental and Comparative Immunology

Gene transcription patterns in response to low level petroleum contaminants in Mytilus trossulus from field sites and harbors in southcentral Alaska

The 1989 Exxon Valdez oil spill damaged a wide range of natural resources, including intertidal communities, and post-spill studies demonstrated acute and chronic exposure and injury to an array of species. Standard toxicological methods to evaluate petroleum contaminants have assessed tissue burdens, with fewer assays providing indicators of health or physiology, particularly when contaminant levels are low and chronic. Marine mussels are a ubiquitous and crucial component of the nearshore environment, and new genomic technologies exist to quantify molecular responses of individual mussels to stimuli, including exposure to polycyclic aromatic hydrocarbons (PAHs). We used gene-based assays of exposure and physiological function to assess chronic oil contamination using the Pacific blue mussel, Mytilus trossulus . We developed a diagnostic gene transcription panel to investigate exposure to PAHs and other contaminants and its effects on mussel physiology and health. During 2012–2015, we analyzed mussels from five field sites in western Prince William Sound, Alaska, with varying oil histories from the 1989 Exxon Valdez oil spill, and from three boat harbors in the area. Gene transcription patterns of mussels from harbors were consistent with elevated exposure to PAHs or other contaminants, whereas transcription patterns of mussels sampled from shorelines in areas affected by the oil spill indicated no PAH exposure.

Alaska

Induction of anti-viral genes during acute infection with Viral hemorrhagic septicemia virus (VHSV) genogroup IVa in Pacific herring ( Clupea pallasii )

Infection with the aquatic rhabdovirus Viral hemorrhagic septicemia virus (VHSV) genogroup IVa results in high mortality in Pacific herring ( Clupea pallasii ) and is hypothesized to be a potential limiting factor for herring recovery. To investigate anti-viral immunity in the Pacific herring, four immune response genes were identified: the myxovirus resistance ( Clpa- Mx), a major histocompatibility complex IB (named Clpa- UAA.001), the inducible immunoproteosome subunit 9 ( Clpa- PSMB9) and the neutrophil chemotactic factor ( Clpa- LECT2). Reverse transcriptase quantitative PCR (RT-qPCR) assays were developed based on these gene sequences to investigate the host immune response to acute VHSV infection following both injection and immersion challenge. Virus levels were measured by both plaque assay and RT-qPCR and peaked at day 6 during the 10-day exposure period for both groups of fish. The interferon stimulated genes ( Clpa- Mx, −UAA.001, and −PSMB9) were significantly up-regulated in response to VHSV infection at both 6 and 10 days post-infection in both spleen and fin. Results from this study indicate that Pacific herring mount a robust, early antiviral response in both fin and spleen tissues. The immunological tools developed in this study will be useful for future studies to investigate antiviral immunity in Pacific herring.

Fish and Shellfish Immunology

Environmental antimicrobial resistance gene detection from wild bird habitats using two methods: A commercially available culture-independent qPCR assay and culture of indicator bacteria followed by whole-genome sequencing

Objectives A variety of methods have been developed to detect antimicrobial resistance (AMR) in different environments to better understand the evolution and dissemination of this public health threat. Comparisons of results generated using different AMR detection methods, such as quantitative PCR (qPCR) and whole-genome sequencing (WGS), are often imperfect, and few studies have analysed samples in parallel to evaluate differences. In this study, we compared bacterial culture and WGS to a culture-independent commercially available qPCR assay to evaluate the concordance between methods and the utility of each in answering research questions regarding the presence and epidemiology of AMR in wild bird habitats. Methods We first assessed AMR gene detection using qPCR in 45 bacterial isolates from which we had existing WGS data. We then analysed 52 wild bird faecal samples and 9 spatiotemporally collected water samples using culture-independent qPCR and WGS of phenotypically resistant indicator bacterial isolates. Results Overall concordance was strong between qPCR and WGS of bacterial isolates, although concordance differed among antibiotic classes. Analysis of wild bird faecal and water samples revealed that more samples were determined to be positive for AMR via qPCR than via culture and WGS of bacterial isolates, although qPCR did not detect AMR genes in two samples from which phenotypically resistant isolates were found. Conclusions Both qPCR and culture followed by sequencing may be effective approaches for characterising AMR genes harboured by wild birds, although data streams produced using these different tools may have advantages and disadvantages that should be considered given the application and sample matrix.

Journal of Global Antimicrobial Resistance

Concurrent speciation in the eastern woodland salamanders (Genus Plethodon):DNA sequences of the complete albumin nuclear and partialmitochondrial 12s genes

Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time.

Molecular Phylogenetics and Evolution

Population genetic structure and gene flow of Adélie penguins (Pygoscelis adeliae) breeding throughout the western Antarctic Peninsula

Adélie penguins ( Pygoscelis adeliae ) are responding to ocean–climate variability throughout the marine ecosystem of the western Antarctic Peninsula (WAP) where some breeding colonies have declined by 80%. Nuclear and mitochondrial DNA (mtDNA) markers were used to understand historical population genetic structure and gene flow given relatively recent and continuing reductions in sea ice habitats and changes in numbers of breeding adults at colonies throughout the WAP. Genetic diversity, spatial genetic structure, genetic signatures of fluctuations in population demography and gene flow were assessed in four regional Adélie penguin colonies. The analyses indicated little genetic structure overall based on bi-parentally inherited microsatellite markers ( F ST =-0.006–0.004). No significant variance was observed in overall haplotype frequency (mtDNA Φ ST =0.017; P =0.112). Some comparisons with Charcot Island were significant, suggestive of female-biased philopatry. Estimates of gene flow based on a two-population coalescent model were asymmetrical from the species’ regional core to its northern range. Breeding Adélie penguins of the WAP are a panmictic population and hold adequate genetic diversity and dispersal capacity to be resilient to environmental change.

Antarctic Science

GeoChip-based analysis of microbial functional gene diversity in a landfill leachate-contaminated aquifer

The functional gene diversity and structure of microbial communities in a shallow landfill leachate-contaminated aquifer were assessed using a comprehensive functional gene array (GeoChip 3.0). Water samples were obtained from eight wells at the same aquifer depth immediately below a municipal landfill or along the predominant downgradient groundwater flowpath. Functional gene richness and diversity immediately below the landfill and the closest well were considerably lower than those in downgradient wells. Mantel tests and canonical correspondence analysis (CCA) suggested that various geochemical parameters had a significant impact on the subsurface microbial community structure. That is, leachate from the unlined landfill impacted the diversity, composition, structure, and functional potential of groundwater microbial communities as a function of groundwater pH, and concentrations of sulfate, ammonia, and dissolved organic carbon (DOC). Historical geochemical records indicate that all sampled wells chronically received leachate, and the increase in microbial diversity as a function of distance from the landfill is consistent with mitigation of the impact of leachate on the groundwater system by natural attenuation mechanisms.

Oklahoma

Bisphenol A and 17α-ethinylestradiol-induced transgenerational gene expression differences in the brain–pituitary–testis axis of medaka, Oryzias latipes

Endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 17α-ethinylestradiol (EE2), can have far reaching health effects, including transgenerational abnormalities in offspring that never directly contacted either chemical. We previously reported reduced fertilization rates and embryo survival at F2 and F3 generations caused by 7-day embryonic exposure (F0) to 100 μg/L BPA or 0.05 μg/L EE2 in medaka. Crossbreeding of fish in F2 generation indicated subfertility in males. To further understand the mechanisms underlying BPA or EE2-induced adult onset and transgenerational reproductive defects in males, the present study examined the expression of genes regulating the brain–pituitary–testis (BPT) axis in the same F0 and F2 generation male medaka. Embryonic exposure to BPA or EE2 led to hyperactivation of brain and pituitary genes, which are actively involved in reproduction in adulthood of the F0 generation male fish, and some of these F0 effects continued to the F2 generation (transgenerational effects). Particularly, the F2 generation inherited the hyperactivated state of expression for kisspeptin ( kiss1 and kiss2 ) and their receptors ( kiss1r and kiss2r), and gnrh and gnrh receptors. At F2 generation, expression of DNA methyltransferase 1 ( dnmt1 ) decreased in brain of the BPA treatment lineage, while EE2 treatment lineage showed increased dnmt3bb expression. Global hypomethylation pattern was observed in the testis of both F0 and F2 generation fish. Taken together, these results demonstrated that BPA or EE2-induced transgenerational reproductive impairment in the F2 generation was associated with alterations of reproductive gene expression in brain and testis and global DNA methylation in testis.

Biology of Reproduction

Gene expression reveals immune response strategies of naïve Hawaiian honeycreepers experimentally infected with introduced avian malaria

The unprecedented rise in the number of new and emerging infectious diseases in the last quarter century poses direct threats to human and wildlife health. The introduction to the Hawaiian archipelago of Plasmodium relictum and the mosquito vector that transmits the parasite has led to dramatic losses in endemic Hawaiian forest bird species. Understanding how mechanisms of disease immunity to avian malaria may evolve is critical as climate change facilitates increased disease transmission to high elevation habitats where malaria transmission has historically been low and the majority of the remaining extant Hawaiian forest bird species now reside. Here, we compare the transcriptomic profiles of highly susceptible Hawai‘i ‘amakihi ( Chlorodrepanis virens ) experimentally infected with P. relictum to those of uninfected control birds from a naïve high elevation population. We examined changes in gene expression profiles at different stages of infection to provide an in-depth characterization of the molecular pathways contributing to survival or mortality in these birds. We show that the timing and magnitude of the innate and adaptive immune response differed substantially between individuals that survived and those that succumbed to infection, and likely contributed to the observed variation in survival. These results lay the foundation for developing gene-based conservation strategies for Hawaiian honeycreepers by identifying candidate genes and cellular pathways involved in the pathogen response that correlate with a bird’s ability to recover from malaria infection.

Hawaii

High inter-population connectivity and occasional gene flow between subspecies improves recovery potential for the endangered Least Bell’s Vireo

Increasingly, genomic data are being used to supplement field-based ecological studies to help evaluate recovery status and trends in endangered species. We collected genomic data to address two related questions regarding the Least Bell’s Vireo ( Vireo bellii ), an endangered migratory songbird restricted to southern California riparian habitat for breeding. First, we sought to delineate the range limits and potential overlap between Least Bell’s Vireo and its sister subspecies, the Arizona Bell’s Vireo, by analyzing samples from the deserts of eastern California, southwestern Nevada, Utah and Arizona. Second, we evaluated genetic structure among Least Bell’s Vireo populations in coastal California and estimated effective population size. Clustering analyses based on 10,571 single nucleotide polymorphisms (SNPs) from 317 samples supported two major groups that aligned closely to the previously defined subspecies ranges. The first cluster included birds in the Central Valley, all coastal drainages, and westernmost deserts of California, with no further sub-structuring among coastal drainages. Almost all birds from the Amargosa River in eastern California and eastward assigned to the second cluster; however, low levels of gene flow were detected across the subspecies groups, with greater rates of gene flow from Arizona Bell’s Vireo to Least Bell’s Vireo than the reverse. Admixed individuals occurred in the California deserts; and although smaller than coastal populations, desert populations may be important for maintaining and replenishing genetic diversity and facilitating the movement of potentially adaptive genes between subspecies. Within Least Bell’s Vireo, local populations in coastal drainages comprised a single genetic population, with some evidence of close relatives distributed across drainages, suggesting these could function as a well-connected metapopulation. These results are consistent with previous Least Bell’s Vireo banding studies that reported high rates of dispersal among drainages. Effective population size for both subspecies was high, suggesting that adaptive potential has been maintained despite previous declines.

Ornithological Applications