Transdermal Measurement of Glomerular Filtration Rate in Preclinical Research.

The measurement of glomerular filtration rate (GFR) is essential to understanding renal physiology, including the monitoring of disease progression and treatment effectiveness. Transdermal measurement of glomerular filtration rate (tGFR) using a miniaturized fluorescence monitor in combination with a fluorescent exogenous GFR tracer has become a common technique to measure GFR in the preclinical setting, especially in rodent models. It allows for close to real-time measurement of GFR in conscious unrestrained animals and overcomes several limitations of other GFR measures. Its widespread use is reflected by published research articles and conference abstracts from different research fields, including in the assessment of new and existing kidney therapeutics, evaluation of nephrotoxicity, screening of novel chemical or medical agents, and fundamental understanding of kidney function.

Sotagliflozin, a Dual SGLT1/2 Inhibitor, Improves Cardiac Outcomes in a Normoglycemic Mouse Model of Cardiac Pressure Overload.

Selective SGLT2 inhibition reduces the risk of worsening heart failure and cardiovascular death in patients with existing heart failure, irrespective of diabetic status. We aimed to investigate the effects of dual SGLT1/2 inhibition, using sotagliflozin, on cardiac outcomes in normal diet (ND) and high fat diet (HFD) mice with cardiac pressure overload. Five-week-old male C57BL/6J mice were randomized to receive a HFD (60% of calories from fat) or remain on ND for 12 weeks. One week later, transverse aortic constriction (TAC) was employed to induce cardiac pressure-overload (50% increase in right:left carotid pressure versus sham surgery), resulting in left ventricular hypertrophic remodeling and cardiac fibrosis, albeit preserved ejection fraction. At 4 weeks post-TAC, mice were treated for 7 weeks by oral gavage once daily with sotagliflozin (10 mg/kg body weight) or vehicle (0.1% tween 80). In ND mice, treatment with sotagliflozin attenuated cardiac hypertrophy and histological markers of cardiac fibrosis induced by TAC. These benefits were associated with profound diuresis and glucosuria, without shifts toward whole-body fatty acid utilization, increased circulating ketones, nor increased cardiac ketolysis. In HFD mice, sotagliflozin reduced the mildly elevated glucose and insulin levels but did not attenuate cardiac injury induced by TAC. HFD mice had vacuolation of proximal tubular cells, associated with less profound sotagliflozin-induced diuresis and glucosuria, which suggests dampened drug action. We demonstrate the utility of dual SGLT1/2 inhibition in treating cardiac injury induced by pressure overload in normoglycemic mice. Its efficacy in high fat-fed mice with mild hyperglycemia and compromised renal morphology requires further study.

Maternal gut microbiota displays minor changes in overweight and obese women with GDM.

BACKGROUND AND AIMS: Previous literature have shown a diversity of findings regarding the relationship between the maternal gut microbiota and gestational diabetes mellitus (GDM). We investigated the gut microbiota of overweight and obese women with gestational diabetes mellitus (GDM) against matched euglycaemic women at 16 and 28-weeks' gestation. METHODS AND RESULTS: This study included women from the SPRING (Study of PRobiotics IN Gestational diabetes) cohort. Overweight and obese women with no impaired glucose tolerance or impaired fasted glucose were enrolled prior to gestational age <16 weeks. Participants with a diagnosis of GDM (n = 29) were matched with euglycaemic (n = 29) women for body mass index, probiotic or placebo intervention, maternal age, parity and ethnicity. Anthropometric, clinical and fecal microbiota (16S rRNA amplicon-based sequencing of V6-V8 region) data was assessed at 16 and 28-weeks' gestation. The relative abundances of key bacterial genera were not significantly altered between euglycaemic women and women with GDM. Occurrence of bacterial taxa was similar between groups at both timepoints. GDM was associated with decreased Shannon diversity (p = 0.02) without differentiated clustering measured by beta diversity at 28-weeks' gestation. CONCLUSIONS: Overweight and obese women with GDM demonstrate minor variation in the gut microbiota at 16 and 28-weeks' gestation compared with matched euglycaemic women. This study expands on previous literature concluding the microbiota does not likely have a disease-specific characterisation in GDM.

Validation of non-invasive transcutaneous measurement for glomerular filtration rate in lean and obese C57BL/6J mice.

AIM: The measurement of glomerular filtration rate (GFR) in experimental rodents is pivotal to understanding the progression of kidney disease and benefits of treatment strategies. A non-invasive clearance device has been developed, which measures transcutaneous decay of injected FITC-sinistrin in conscious rodents. The technique was validated against the well-established plasma clearance method in the same mice, but on consecutive days, using only models of uninephrectomy and polycystic kidney disease. We aimed to validate this widely used technique in the same lean or obese mice, at the same time. METHODS: Five-week-old male C57BL/6J mice were randomised to a high fat diet (n = 12) or normal diet (n = 11) for 10 weeks. Transcutaneous and plasma clearance of FITC-sinistrin were measured simultaneously in each mouse. RESULTS: In lean mice, there was a positive correlation between transcutaneous and plasma derived GFR (P < .01, R2 = .704), although there was an approximate 40% underestimation by the transcutaneous method (P < .0001). In obese mice, no correlation was observed between transcutaneous and plasma derived GFR, nor elimination half-life which removes any effect of the conversion factor and injected dose. The limits of agreement in a Bland-Altman plot were narrower when we used new conversion factors derived from mice in the current study and, in lean mice, a generic conversion factor which assumes 20% extracellular volume. CONCLUSION: The non-invasive clearance device may be useful for serial GFR measurements in lean and healthy mice, provided validation studies have been carried out, but its utility in obesity requires further study.

Changes in genetic diversity and differentiation in Red-cockaded woodpeckers (Dryobates borealis) over the past century.

Red-cockaded woodpeckers (RCW; Dryobates borealis) declined after human activities reduced their fire-maintained pine ecosystem to <3% of its historical range in the southeastern United States and degraded remaining habitat. An estimated 1.6 million RCW cooperative breeding groups declined to about 3,500 groups with no more than 10,000 birds by 1978. Management has increased RCW population abundances since they were at their lowest in the 1990s. However, no range-wide study has been undertaken since then to investigate the impacts of this massive bottleneck or infer the effects of conservation management and recent demographic recoveries. We used mitochondrial DNA sequences (mtDNA) and nine nuclear microsatellite loci to determine if range-wide demographic declines resulted in changes to genetic structure and diversity in RCW by comparing samples collected before 1970 (mtDNA data only), between 1992 and 1995 (mtDNA and microsatellites), and between 2010 and 2014 (mtDNA and microsatellites). We show that genetic diversity has been lost as detected by a reduction in the number of mitochondrial haplotypes. This reduction was apparent in comparisons of pre-1970 mtDNA data with data from the 1992-1995 and 2010-2014 time points, with no change between the latter two time points in mtDNA and microsatellite analyses. The mtDNA data also revealed increases in range-wide genetic differentiation, with a genetically panmictic population present throughout the southeastern United States in the pre-1970s data and subsequent development of genetic structure that has remained unchanged since the 1990s. Genetic structure was also uncovered with the microsatellite data, which like the mtDNA data showed little change between the 1992-1995 and 2010-2014 data sets. Temporal haplotype networks revealed a consistent, star-like phylogeny, suggesting that despite the overall loss of haplotypes, no phylogenetically distinct mtDNA lineages were lost when the population declined. Our results may suggest that management during the last two decades has prevented additional losses of genetic diversity.

Isolation by distance versus landscape resistance: Understanding dominant patterns of genetic structure in Northern Spotted Owls (Strix occidentalis caurina).

Landscape genetics investigations examine how the availability and configuration of habitat influence genetic structure of plants and animals. We used landscape genetics to evaluate the role that forest connectivity plays in determining genetic structure of the federally-threatened Northern Spotted Owl (Strix occidentalis caurina) using genotypes of 339 Northern Spotted Owls obtained for 10 microsatellite loci. Spatial clustering analyses identified a distinct genetic cluster at the southern extent of the region examined. This cluster could not be linked to landscape connectivity patterns and suggested that post-Pleistocene processes were involved with its development rather than contemporary landscape configuration. We also compared matrices of pairwise inter-individual genetic distances with resistance distances derived from a circuit-theory based framework. Resistance distances were obtained for an idealized raster map that reflected continuous unimpeded dispersal habitat across the landscape along with five empirically-derived raster maps reflecting the 1870's, 1940's, 1986, 1994, and 2012. Resistance distances from the idealized map served as surrogates for linear geographic distances. Relative to idealized conditions, resistance distances were ~250% higher in the 1940's and ~200% higher from 1986 onward. Resistance distances from the 1870's were ~40% higher than idealized conditions. Inter-individual genetic distances were most highly correlated with resistance distances from the idealized map rather than any of the empirical maps. Two hypotheses explain our results. First, our results may reflect temporal lags between the onset of large-scale habitat alterations and their novel effects on genetic structure in long-lived species such as Northern Spotted Owls. Second, because Northern Spotted Owls disperse over long distances, our results may indicate that forest habitat has never been sufficiently fragmented to the point where connectivity was disrupted. The second hypothesis could indicate that forest management practices mandated by the Northwest Forest Plan succeeded with one of its primary goals. However, our results do not represent a complete portrayal of the status of Northern Spotted Owls given detection of significant population declines and bottlenecks in other studies. Future investigations based on computer simulations may help distinguish between hypotheses.

Modeling heart failure risk in diabetes and kidney disease: limitations and potential applications of transverse aortic constriction in high-fat-fed mice.

There is an increased incidence of heart failure in individuals with diabetes mellitus (DM). The coexistence of kidney disease in DM exacerbates the cardiovascular prognosis. Researchers have attempted to combine the critical features of heart failure, using transverse aortic constriction, with DM in mice, but variable findings have been reported. Furthermore, kidney outcomes have not been assessed in this setting; thus its utility as a model of heart failure in DM and kidney disease is unknown. We generated a mouse model of obesity, hyperglycemia, and mild kidney pathology by feeding male C57BL/6J mice a high-fat diet (HFD). Cardiac pressure overload was surgically induced using transverse aortic constriction (TAC). Normal diet (ND) and sham controls were included. Heart failure risk factors were evident at 8-wk post-TAC, including increased left ventricular mass (+49% in ND and +35% in HFD), cardiomyocyte hypertrophy (+40% in ND and +28% in HFD), and interstitial and perivascular fibrosis (Masson's trichrome and picrosirius red positivity). High-fat feeding did not exacerbate the TAC-induced cardiac outcomes. At 11 wk post-TAC in a separate mouse cohort, echocardiography revealed reduced left ventricular size and increased left ventricular wall thickness, the latter being evident in ND mice only. Systolic function was preserved in the TAC mice and was similar between ND and HFD. Thus combined high-fat feeding and TAC in mice did not model the increased incidence of heart failure in DM patients. This model, however, may mimic the better cardiovascular prognosis seen in overweight and obese heart failure patients.

Genetic differentiation and inferred dynamics of a hybrid zone between Northern Spotted Owls (Strix occidentalis caurina) and California Spotted Owls (S. o. occidentalis) in northern California.

Genetic differentiation among Spotted Owl (Strix occidentalis) subspecies has been established in prior studies. These investigations also provided evidence for introgression and hybridization among taxa but were limited by a lack of samples from geographic regions where subspecies came into close contact. We analyzed new sets of samples from Northern Spotted Owls (NSO: S. o. caurina) and California Spotted Owls (CSO: S. o. occidentalis) in northern California using mitochondrial DNA sequences (mtDNA) and 10 nuclear microsatellite loci to obtain a clearer depiction of genetic differentiation and hybridization in the region. Our analyses revealed that a NSO population close to the northern edge of the CSO range in northern California (the NSO Contact Zone population) is highly differentiated relative to other NSO populations throughout the remainder of their range. Phylogenetic analyses identified a unique lineage of mtDNA in the NSO Contact Zone, and Bayesian clustering analyses of the microsatellite data identified the Contact Zone as a third distinct population that is differentiated from CSO and NSO found in the remainder of the subspecies' range. Hybridization between NSO and CSO was readily detected in the NSO Contact Zone, with over 50% of individuals showing evidence of hybrid ancestry. Hybridization was also identified among 14% of CSO samples, which were dispersed across the subspecies' range in the Sierra Nevada Mountains. The asymmetry of hybridization suggested that the hybrid zone may be dynamic and moving. Although evidence of hybridization existed, we identified no F1 generation hybrid individuals. We instead found evidence for F2 or backcrossed individuals among our samples. The absence of F1 hybrids may indicate that (1) our 10 microsatellites were unable to distinguish hybrid types, (2) primary interactions between subspecies are occurring elsewhere on the landscape, or (3) dispersal between the subspecies' ranges is reduced relative to historical levels, potentially as a consequence of recent regional fires.

Hello Harlie: Enabling Speech Monitoring Through Chat-Bot Conversations.

People with neurological conditions such as Parkinson's disease and dementia are known to have difficulties in language and communication. This paper presents initial testing of an artificial conversational agent, called Harlie. Harlie runs on a smartphone and is able to converse with the user on a variety of topics. A description of the application and a sample dialog are provided to illustrate the various roles chat-bots can play in the management of neurological conditions. Harlie can be used for measuring voice and communication outcomes during the daily life of the user, and for gaining information about challenges encountered. Moreover, it is anticipated that she may also have an educational and support role.

The conservation genetics juggling act: integrating genetics and ecology, science and policy.

The field of conservation genetics, when properly implemented, is a constant juggling act integrating molecular genetics, ecology, and demography with applied aspects concerning managing declining species or implementing conservation laws and policies. This young field has grown substantially since the 1980s following the development of polymerase chain reaction and now into the genomics era. Our laboratory has 'grown up' with the field, having worked on these issues for over three decades. Our multidisciplinary approach entails understanding the behavior and ecology of species as well as the underlying processes that contribute to genetic viability. Taking this holistic approach provides a comprehensive understanding of factors that influence species persistence and evolutionary potential while considering annual challenges that occur throughout their life cycle. As a federal laboratory, we are often addressing the needs of the U.S. Fish and Wildlife Service in their efforts to list, de-list, or recover species. Nevertheless, there remains an overall communication gap between research geneticists and biologists who are charged with implementing their results. Therefore, we outline the need for a National Center for Small Population Biology to ameliorate this problem and provide organizations charged with making status decisions firmer ground from which to make their critical decisions.

Intercontinental genetic structure and gene flow in Dunlin (Calidris alpina), a potential vector of avian influenza.

Waterfowl (Anseriformes) and shorebirds (Charadriiformes) are the most common wild vectors of influenza A viruses. Due to their migratory behavior, some may transmit disease over long distances. Migratory connectivity studies can link breeding and nonbreeding grounds while illustrating potential interactions among populations that may spread diseases. We investigated Dunlin (Calidris alpina), a shorebird with a subspecies (C. a. arcticola) that migrates from nonbreeding areas endemic to avian influenza in eastern Asia to breeding grounds in northern Alaska. Using microsatellites and mitochondrial DNA, we illustrate genetic structure among six subspecies: C. a. arcticola,C. a. pacifica,C. a. hudsonia,C. a. sakhalina,C. a. kistchinski, and C. a. actites. We demonstrate that mitochondrial DNA can help distinguish C. a. arcticola on the Asian nonbreeding grounds with >70% accuracy depending on their relative abundance, indicating that genetics can help determine whether C. a. arcticola occurs where they may be exposed to highly pathogenic avian influenza (HPAI) during outbreaks. Our data reveal asymmetric intercontinental gene flow, with some C. a. arcticola short-stopping migration to breed with C. a. pacifica in western Alaska. Because C. a. pacifica migrates along the Pacific Coast of North America, interactions between these subspecies and other taxa provide route for transmission of HPAI into other parts of North America.

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

Variation in migratory behavior influences regional genetic diversity and structure among American Kestrel populations (Falco sparverius) in North America.

Birds employ numerous strategies to cope with seasonal fluctuations in high-quality habitat availability. Long distance migration is a common tactic; however, partial migration is especially common among broadly distributed species. Under partial migration systems, a portion of a species migrates, whereas the remainder inhabits breeding grounds year round. In this study, we identified effects of migratory behavior variation on genetic structure and diversity of American Kestrels (Falco sparverius), a widespread partial migrant in North America. American Kestrels generally migrate; however, a resident group inhabits the southeastern United States year round. The southeastern group is designated as a separate subspecies (F. s. paulus) from the migratory group (F. s. sparverius). Using mitochondrial DNA and microsatellites from 183 and 211 individuals, respectively, we illustrate that genetic structure is stronger among nonmigratory populations, with differentiation measures ranging from 0.060 to 0.189 depending on genetic marker and analysis approach. In contrast, measures from western North American populations ranged from 0 to 0.032. These findings suggest that seasonal migratory behavior is also associated with natal and breeding dispersal tendencies. We likewise detected significantly lower genetic diversity within nonmigratory populations, reflecting the greater influence of genetic drift in small populations. We identified the signal of population expansion among nonmigratory populations, consistent with the recent establishment of higher latitude breeding locations following Pleistocene glacial retreat. Differentiation of F. s. paulus and F. s. sparverius reflected subtle differences in allele frequencies. Because migratory behavior can evolve quickly, our analyses suggest recent origins of migratory American Kestrel populations in North America.

Range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) in northwestern North America.

The dynamic geological and climatic history of northwestern North America has made it a focal region for phylogeography. We conducted a range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) across its range in northwestern North America to understand its evolutionary history and the distribution of clades to inform conservation of R. pretiosa and Great Basin R. luteiventris, candidates for listing under the US Endangered Species Act. Mitochondrial DNA sequence data from a segment of the cytochrome b gene were obtained from 308 R. luteiventris and R. pretiosa from 96 sites. Phylogenetic analysis revealed one main R. pretiosa clade and three main R. luteiventris clades, two of which overlapped in southeastern Oregon. The three R. luteiventris clades were separated from each other by high levels of sequence divergence (average of 4.75-4.97%). Two divergent clades were also uncovered within the Great Basin. Low genetic variation in R. pretiosa and the southeastern Oregon clade of R. luteiventris suggests concern about their vulnerability to extinction.

Introgression and dispersal among spotted owl (Strix occidentalis) subspecies.

Population genetics plays an increasingly important role in the conservation and management of declining species, particularly for defining taxonomic units. Subspecies are recognized by several conservation organizations and countries and receive legal protection under the US Endangered Species Act (ESA). Two subspecies of spotted owls, northern (Strix occidentalis caurina) and Mexican (S. o. lucida) spotted owls, are ESA-listed as threatened, but the California (S. o. occidentalis) spotted owl is not listed. Thus, determining the boundaries of these subspecies is critical for effective enforcement of the ESA. We tested the validity of previously recognized spotted owl subspecies by analysing 394 spotted owls at 10 microsatellite loci. We also tested whether northern and California spotted owls hybridize as suggested by previous mitochondrial DNA studies. Our results supported current recognition of three subspecies. We also found bi-directional hybridization and dispersal between northern and California spotted owls centered in southern Oregon and northern California. Surprisingly, we also detected introgression of Mexican spotted owls into the range of northern spotted owls, primarily in the northern part of the subspecies' range in Washington, indicating long-distance dispersal of Mexican spotted owls. We conclude with a discussion of the conservation implications of our study.

Taxonomic considerations in listing subspecies under the U.S. Endangered Species Act.

The U.S. Endangered Species Act (ESA) allows listing of subspecies and other groupings below the rank of species. This provides the U.S. Fish and Wildlife Service and the National Marine Fisheries Service with a means to target the most critical unit in need of conservation. Although roughly one-quarter of listed taxa are subspecies, these management agencies are hindered by uncertainties about taxonomic standards during listing or delisting activities. In a review of taxonomic publications and societies, we found few subspecies lists and none that stated standardized criteria for determining subspecific taxa. Lack of criteria is attributed to a centuries-old debate over species and subspecies concepts. Nevertheless, the critical need to resolve this debate for ESA listings led us to propose that minimal biological criteria to define disjunct subspecies (legally or taxonomically) should include the discreteness and significance criteria of distinct population segments (as defined under the ESA). Our subspecies criteria are in stark contrast to that proposed by supporters of the phylogenetic species concept and provide a clear distinction between species and subspecies. Efforts to eliminate or reduce ambiguity associated with subspecies-level classifications will assist with ESA listing decisions. Thus, we urge professional taxonomic societies to publish and periodically update peer-reviewed species and subspecies lists. This effort must be paralleled throughout the world for efficient taxonomic conservation to take place.