Paper spray mass spectrometry combined with machine learning as a rapid diagnostic for chronic kidney disease.

A new analytical method for chronic kidney disease (CKD) detection utilizing paper spray mass spectrometry (PS-MS) combined with machine learning is presented. The analytical protocol is rapid and simple, based on metabolic profile alterations in urine. Anonymized raw urine samples were deposited (10 µL each) onto pointed PS-MS sample strips. Without waiting for the sample to dry, 75 µL of acetonitrile and high voltage were applied to the strips, using high resolution mass spectrometry measurement (15 s per sample) with polarity switching to detect a wide range of metabolites. Random forest machine learning was used to classify the resulting data. The diagnostic performance for the potential diagnosis of CKD was evaluated for accuracy, sensitivity, and specificity, achieving results >96% for the training data and >91% for validation and test data sets. Metabolites selected by the classification model as up- or down-regulated in healthy or CKD samples were tentatively identified and in agreement with previously reported literature. The potential utilization of this approach to discriminate albuminuria categories (normo, micro, and macroalbuminuria) was also demonstrated. This study indicates that PS-MS combined with machine learning has the potential to be used as a rapid and simple diagnostic tool for CKD.

Project-specific bumble bee habitat quality assessment.

The listing of Bombus affinis Cresson 1863 (Rusty Patched Bumble Bee; RPBB) in 2017 under the Endangered Species Act (ESA) created a regulatory need for assessment methods, in order to limit take of this species by construction and development. As the first social insect listed under the ESA, the listing of RPBB has required new methods for biological assessment. This species has a complex life cycle requiring a mosaic of different habitat types, with each life cycle stage facing unique challenges and threats. I have established a method for separately assessing habitats critical to each vulnerable life history stage, using a combination of aerial photography, GIS maps and target-specific ground survey efforts. This method identifies factors that may potentially limit bumble bee colony success in each stage and provides project planners with facts about physical structures or plant communities that may have elevated importance to bumble bees during certain seasonal windows. Previous efforts to assess bumble bee habitat considered landscape features thought to be linked to bumble bee colony success. This effort extends these methods to estimate project specific impacts of construction and development projects, necessary for Section 7 Consultation with the United States Fish and Wildlife Service (USFWS) under the ESA. •Systematic spatial assessment of landscape features linked to critical periods in the life history trajectory of a bumble bee colony across a season•Construction and development project proponents can approach USFWS consultation with quantitative estimates of the area of a project area classified by habitat types and qualities•Factors limiting bumble bee recovery may be inferred from the distribution and abundance of the constituent elements of quality bumble bee habitat.

Simultaneous quantitation of urinary albumin and creatinine for rapid clinical albuminuria diagnostics using high-throughput paper spray mass spectrometry.

Albuminuria is a clinical condition associated with poor kidney function, diagnosed by determining the ratio of albumin to creatinine concentrations in patient urine samples. We present a high-throughput paper spray mass spectrometry (PS-MS) method for simultaneous quantitation of urinary albumin and creatinine for potential diagnosis of albuminuria. Minimal (urine dilution) or no sample preparation is required. The analytical performance of the method was evaluated, achieving linear calibration curves (R2 > 0.99) with little inter-day variability in the slope (N = 5 days), exhibiting coefficient of variation (CV) of 8% and 3% for albumin and creatinine, respectively. LOD and LOQ for albumin were 2.1 and 7.0 mg L-1, and for creatinine were 0.01 and 0.03 mmol L-1, respectively. Intra- and inter-day (N = 5) precisions (%CV) and accuracies (%bias) were <10% and ±11%, respectively, for both analytes. The method was applied to determine albumin-to-creatinine ratios in anonymous human patient urine samples (N = 56), and a correlation of R2 = 0.9744 was achieved between the PS-MS results and validated clinical method results. This work demonstrates the utility of PS-MS to simultaneously quantify a large (albumin) and a small (creatinine) molecule directly in patient urine samples, and its potential as a tool for clinical albuminuria diagnostics.

Canadian SARS-CoV-2 serological survey using antenatal serum samples: a retrospective seroprevalence study.

BACKGROUND: Insufficient data on the rate and distribution of SARS-CoV-2 infection in Canada has presented a substantial challenge to the public health response to the COVID-19 pandemic. Our objective was to assess SARS-CoV-2 seroprevalence in a representative sample of pregnant people throughout Canada, across multiple time points over 2 years of the pandemic, to describe the seroprevalence and show the ability of this process to provide prevalence estimates. METHODS: This Canadian retrospective serological surveillance study used existing serological prenatal samples across 10 provinces over multiple time periods: Feb. 3-21, 2020; Aug. 24-Sept. 11, 2020; Nov. 16-Dec. 4, 2020; Nov. 15-Dec. 3, 2021; and results from the province of British Columbia during a period in which the SARS-CoV-2 B.1.1.529 (Omicron) variant was predominant, from Nov. 15, 2021, to June 11, 2022. Age and postal code administrative data allowed for comparison with concurrent polymerase chain reactivity (PCR)-positive results collected by Statistics Canada and the Canadian Surveillance of COVID-19 in Pregnancy (CANCOVID-Preg) project. RESULTS: Seropositivity in antenatal serum as early as February 2020 indicates SARS-CoV-2 transmission before the World Health Organization's declaration of the pandemic. Seroprevalence in our sample of pregnant people was 1.84 to 8.90 times higher than the recorded concurrent PCR-positive prevalence recorded among females aged 20-49 years in November-December 2020. Overall seropositivity in our sample of pregnant people was low at the end of 2020, increasing to 15% in 1 province by the end of 2021. Seroprevalence among pregnant people in BC during the Omicron period increased from 5.8% to 43% from November 2021 to June 2022. INTERPRETATION: These results indicate widespread vulnerability to SARS-CoV-2 infection before vaccine availability in Canada. During the time periods sampled, public health tracking systems were under-reporting infections, and seroprevalence results during the Omicron period indicate extensive community spread of SARS-CoV-2 infection.

Evaluation of the stability of fecal immunochemical test specimens.

Fecal immunochemical tests (FIT) are used to screen for colorectal cancer by detecting blood present in stool. Patients collect FIT specimens at home in a sampling kit and return them to the lab for testing. At our institution, patients are instructed to return their specimens to the laboratory within seven days from collection, which is shorter than the manufacturer stated room temperature (RT) stability of 15 days. The objective of this study was to assess and verify the stability of FIT specimens at RT and to determine if refrigerated storage improves stability. A series of experiments were performed with the OC-Sensor DIANA iFOB Test system between 2017 and 2019, using a positive clinical cut-off of 75 ng/mL (15 µg/g) hemoglobin (Hb). Specimens were collected and categorized based on their initially measured Hb concentration and had repeated measurements for up to 21 days following collection. FIT specimens were stored either at RT or refrigerated. Our results show that FIT specimens have reduced concentrations of Hb compared to baseline when stored at RT; refrigeration improved FIT specimen stability but did not completely prevent the reduction in Hb concentration. Additionally, specimens marginally above the cut-off (initial concentrations between 75 and 100 ng/mL (15-20 µg/g)) that were stored at RT showed 100% positivity on the day of collection (n=33), 63% on Day 3 (n=19), 46% on Days 4/5 (n=26), and 38% on Days 6/7 (n=26). Finally, specimens with Hb values near the clinical cut-off appear to be particularly susceptible to false negatives as a result of the reduction in Hb over time. Therefore, laboratories should verify the specifics of their FIT tests before offering it to patients to reduce false negatives.

Canadian society of clinical chemists (CSCC) interim consensus guidance for testing and reporting of SARS-CoV-2 serology.

Clinical laboratories across the world are working to validate and perform testing for SARS-CoV-2 antibodies. Herein, we present interim consensus guidance for Canadian clinical laboratories testing and reporting SARS-CoV-2 serology, with emphasis on the capabilities and limitations of these tests and recommendations for interpretative comments in an effort to achieve harmonized laboratory practices. The consensus document provides a broad overview of topics including sample type and contamination risk; kinetics of antibody response to COVID-19 and the impact on serology testing; clinical utility of SARS-CoV-2 serology testing; clinical performance of commercial laboratory-based assays commonly deployed in North America; recommendations for interim reporting; utility of SARS-CoV-2 antibody testing for pediatric patients; and utility of point-of-care testing. The information is based on the current literature and is subject to change as additional information becomes available.

Effects of supplemental creatine and guanidinoacetic acid on spatial memory and the brain of weaned Yucatan miniature pigs.

The emergence of creatine as a potential cognitive enhancement supplement for humans prompted an investigation as to whether supplemental creatine could enhance spatial memory in young swine. We assessed memory performance and brain concentrations of creatine and its precursor guanidinoacetic acid (GAA) in 14-16-week-old male Yucatan miniature pigs supplemented for 2 weeks with either 200 mg/kg∙d creatine (+Cr; n = 7) or equimolar GAA (157 mg/kg∙d) (+GAA; n = 8) compared to controls (n = 14). Spatial memory tests had pigs explore distinct sets of objects for 5 min. Objects were spatially controlled, and we assessed exploration times of previously viewed objects relative to novel objects in familiar or novel locations. There was no effect of either supplementation on memory performance, but pigs successfully identified novel objects after 10 (p < 0.01) and 20 min (p < 0.01) retention intervals. Moreover, pigs recognized spatial transfers after 65 min (p < 0.05). Regression analyses identified associations between the ability to identify novel objects in memory tests and concentrations of creatine and GAA in cerebellum, and GAA in prefrontal cortex (p < 0.05). The concentration of creatine in brain regions was not influenced by creatine supplementation, but GAA supplementation increased GAA concentration in cerebellum (p < 0.05), and the prefrontal cortex of +GAA pigs had more creatine/g and less GAA/g compared to +Cr pigs (p < 0.05). Creatine kinase activity and maximal reaction velocity were also higher with GAA supplementation in prefrontal cortex (p < 0.05). In conclusion, there appears to be a relationship between memory performance and guanidino compounds in the cerebellum and prefrontal cortex, but the effects were unrelated to dietary supplementation. The cerebellum is identified as a target site for GAA accretion.

Rising above helium: A hydrogen carrier gas chromatography flame ionization detection (GC-FID) method for the simultaneous quantification of toxic alcohols and ethylene glycol in human plasma specimens.

Here we validate a GC, Flame Ionization Detection (GC-FID), liquid injection method using hydrogen as a carrier gas combining analysis of toxic volatile alcohols (VA): methanol, ethanol, isopropanol, acetone, as well as glycols, ethylene glycol (EG) and propylene glycol (PG), in a single method. METHODOLOGY: 200 µL of calibrator, QC, or patient specimen were deproteinized with 400 µL of acetonitrile containing internal standards (10 mmol/L N-propyl alcohol for VA and 2.5 mmol/L 1,2-butanediol for glycols). GC-FID analysis using hydrogen carrier gas and nitrogen makeup gas utilized an Agilent 7890 system equipped with Agilent 7683 liquid autosampler on a 30 m × 530 µm RTX-200 fused silica column. Method validation included repeatability, recovery, carryover, linearity, lower limit of quantification (LLOQ), accuracy, selectivity and measurement uncertainty. RESULTS: The 8.3 min from injection to injection reduced time of analysis by 45% over a previously reported method using Helium carrier gas with no loss in resolution. Within-run and Between-run variability were ≤1.4% and ≤6.8% respectively. Recovery was 100% within a 95% confidence interval. Carryover was negligible for all but EG. LLOQ was <1 mmol/L for all analytes. The upper range of linearity was 120 mmol/L for methanol, ethanol and isopropanol, 100 mmol/L for acetone and 50 mmol/L for EG. Analytes demonstrated acceptable accuracy and measurement uncertainty using College of American Pathologists (CAP) criteria. Toluene can cause a false positive EG, while benzene, xylene and 1,3 butanediol can cause false negative EG. CONCLUSIONS: Converting from Helium to Hydrogen carrier gas benefits patient care through a reduction in turnaround time and provides a cost savings to the laboratory.

Urine protein detection by dipstick: No interference from alkalinity or specific gravity.

Tetrabromphenol blue dye based methods are used to detect proteinuria using urinalysis dipsticks. Manufacturers have claimed that alkalinity leads to false positive proteinuria, and that high specific gravity leads to false negative protein results. However, published reports describing this phenomenon remain equivocal. This study aimed to determine whether pH and/or specific gravity affect protein detection in patient urine using three different tetrabromophenol blue dye-based dipsticks. Patient urine pools were divided into individual aliquots with varied pH or specific gravity, and measured for protein in triplicate using iChem 10SG, iChem Velocity, and Multistix 8SG dipsticks. The pH experiment involved progressive alkalinization of urine aliquots with either 1M NaOH, Na2CO3, or NaHCO3; pH was recorded by electrode. The specific gravity experiment involved mixing aliquots with NaCl and spiking with human albumin. Urine electrolytes and total CO2 were measured (Roche cobas 8000). Fresh patient urines (N = 35) were analyzed for physiological urine pH and total CO2. Urine protein results were not affected by NaOH alkalinization up to pH 10.9. False positive protein occurred at pH 9.9 and >97 mmol/L total CO2 (Na2CO3 alkalization; P < .05). Moreover, false positive protein occurred at pH 7.6 when total CO2 exceeded 137 mmol/L (NaHCO3 alkalization; P < .05). Fresh patient urines did not exceed pH 8.5 or 86 mmol/L total CO2. NaCl elevated specific gravity and caused false negative protein detection when urine ionic strength was >1100 mmol/L (P < .05). Tetrabromphenol blue dipsticks provide robust detection of proteinuria when human urine is within physiological pH, total CO2 and ionic strength.

Prematurity reduces citrulline-arginine-nitric oxide production and precedes the onset of necrotizing enterocolitis in piglets.

Necrotizing enterocolitis (NEC) is associated with low plasma arginine and vascular dysfunction. It is not clear whether low intestinal citrulline production, the precursor for arginine synthesis, occurs before and thus predisposes to NEC or if it results from tissue damage. This study was designed to test the hypothesis that whole body rates of citrulline, arginine, and nitric oxide synthesis are low in premature pigs and that they precede NEC. Piglets delivered by cesarean section at 103 days [preterm (PT)], 110 days [near-term (NT)], or 114 days [full-term (FT)] of gestation were given total parenteral nutrition and after 2 days orogastrically fed infant formula for 42 h to induce NEC. Citrulline and arginine fluxes were determined before and during the feeding protocol. Gross macroscopic and histological NEC scores and plasma fatty acid binding protein (iFABP) concentration were determined as indicators of NEC. Intestinal gene expression for enzymes of the arginine pathway were quantitated. A lower ( P < 0.05) survival rate was observed for PT (8/27) than for NT (9/9) and FT pigs (11/11). PT pigs had higher macroscopic gross ( P < 0.05) and histological NEC ( P < 0.05) scores and iFABP concentration ( P < 0.05) than pigs of more advanced gestational age. PT pigs had lower citrulline production and arginine fluxes ( P < 0.05) throughout and a reduced gene expression in genes of the citrulline-arginine pathway. In summary, intestinal enzyme expression and whole body citrulline and arginine fluxes were reduced in PT pigs compared with animals of more advance gestational age and preceded the development of NEC. NEW & NOTEWORTHY Arginine supplementation prevents necrotizing enterocolitis (NEC), the most common gastrointestinal emergency of prematurity. Citrulline (precursor for arginine) production is reduced during NEC, and this is believed to be a consequence of intestinal damage. In a swine model of NEC, we show that intestinal gene expression of the enzymes for citrulline production and whole body citrulline and arginine fluxes are reduced and precede the onset of NEC in premature pigs. Reduced citrulline production during prematurity may be a predisposition to NEC.

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates.

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4-8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [13C1]methionine and [2H3-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 µg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70-80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.

A fossil caddisfly (Insecta: Trichoptera) from the Eocene of Colorado.

A new genus and species of fossil caddisfly (Insecta: Trichoptera) from the Lower Eocene (Ypresian) Green River Formation of Colorado is described. Litholimnephilops yinani gen. et sp. nov. is the first adult caddisfly to be described from the Green River Formation, and is characterized by large adult body size, presence of ocelli, dark leg spines, and a lack of terminal crossveins in the anterior anastomosis region of the forewings. Terminal genitalia are not visible in the preserved specimen. Familial placement is uncertain, though similarities with the families Limnephilidae and Phryganeidae are observed.

The intestinal-renal axis for arginine synthesis is present and functional in the neonatal pig.

The intestinal-renal axis for endogenous arginine synthesis is an interorgan process in which citrulline produced in the small intestine is utilized by the kidney for arginine synthesis. The function of this axis in neonates has been questioned because during this period the enzymes needed for arginine synthesis argininosuccinate synthase (ASS1) and lyase (ASL) are present in the gut. However, evidence of high plasma citrulline concentrations in neonates suggests otherwise. We quantified in vivo citrulline production in premature (10 days preterm), neonatal (7 days old), and young pigs (35 days old) using citrulline tracers. Neonatal pigs had higher fluxes (69 µmol·kg-1·h-1, P < 0.001) than premature and young pigs (43 and 45 µmol·kg-1·h-1, respectively). Plasma citrulline concentration was also greater in neonatal pigs than in the other age groups. We also determined the site of synthesis and utilization of citrulline in neonatal and young pigs by measuring organ balances across the gut and the kidney. Citrulline was released from the gut and utilized by the kidney in both neonatal and young pigs. The abundance and localization of the enzymes involved in the synthesis and utilization were determined in intestinal and kidney tissue. Despite the presence of ASS1 and ASL in the neonatal small intestine, the lack of colocalization with the enzymes that produce citrulline results in the release of citrulline by the PDV and its utilization by the kidney to produce arginine. In conclusion, the intestinal-renal axis for arginine synthesis is present in the neonatal pig.

Species-free species distribution models describe macroecological properties of protected area networks.

Among the greatest challenges facing the conservation of plants and animal species in protected areas are threats from a rapidly changing climate. An altered climate creates both challenges and opportunities for improving the management of protected areas in networks. Increasingly, quantitative tools like species distribution modeling are used to assess the performance of protected areas and predict potential responses to changing climates for groups of species, within a predictive framework. At larger geographic domains and scales, protected area network units have spatial geoclimatic properties that can be described in the gap analysis typically used to measure or aggregate the geographic distributions of species (stacked species distribution models, or S-SDM). We extend the use of species distribution modeling techniques in order to model the climate envelope (or "footprint") of individual protected areas within a network of protected areas distributed across the 48 conterminous United States and managed by the US National Park System. In our approach we treat each protected area as the geographic range of a hypothetical endemic species, then use MaxEnt and 5 uncorrelated BioClim variables to model the geographic distribution of the climatic envelope associated with each protected area unit (modeling the geographic area of park units as the range of a species). We describe the individual and aggregated climate envelopes predicted by a large network of 163 protected areas and briefly illustrate how macroecological measures of geodiversity can be derived from our analysis of the landscape ecological context of protected areas. To estimate trajectories of change in the temporal distribution of climatic features within a protected area network, we projected the climate envelopes of protected areas in current conditions onto a dataset of predicted future climatic conditions. Our results suggest that the climate envelopes of some parks may be locally unique or have narrow geographic distributions, and are thus prone to future shifts away from the climatic conditions in these parks in current climates. In other cases, some parks are broadly similar to large geographic regions surrounding the park or have climatic envelopes that may persist into near-term climate change. Larger parks predict larger climatic envelopes, in current conditions, but on average the predicted area of climate envelopes are smaller in our single future conditions scenario. Individual units in a protected area network may vary in the potential for climate adaptation, and adaptive management strategies for the network should account for the landscape contexts of the geodiversity or climate diversity within individual units. Conservation strategies, including maintaining connectivity, assessing the feasibility of assisted migration and other landscape restoration or enhancements can be optimized using analysis methods to assess the spatial properties of protected area networks in biogeographic and macroecological contexts.

Protein Synthesis in Mucin-Producing Tissues Is Conserved When Dietary Threonine Is Limiting in Piglets.

BACKGROUND: The neonatal gastrointestinal tract extracts the majority of dietary threonine on the first pass to maintain synthesis of threonine-rich mucins in mucus. As dietary threonine becomes limiting, this extraction must limit protein synthesis in extraintestinal tissues at the expense of maintaining protein synthesis in mucin-producing tissues. OBJECTIVE: The objective was to determine the dietary threonine concentration at which protein synthesis is reduced in various tissues. METHODS: Twenty Yucatan miniature piglets (10 females; mean ± SD age, 15 ± 1 d; mean ± SD weight, 3.14 ± 0.30 kg) were fed 20 test diets with different threonine concentrations, from 0.5 to 6.0 g/100 g total amino acids (AAs; i.e., 20-220% of requirement), and various tissues were analyzed for protein synthesis by administering a flooding dose of [3H]phenylalanine. The whole-body requirement was determined by [1-14C]phenylalanine oxidation and plasma threonine concentrations. RESULTS: Breakpoint analysis indicated a whole-body requirement of 2.8-3.0 g threonine/100 g total AAs. For all of the non-mucin-producing tissues as well as lung and colon, breakpoint analyses indicated decreasing protein synthesis rates below the following concentrations (expressed in g threonine/100 g total AAs; mean ± SE): gastrocnemius muscle, 1.76 ± 0.23; longissimus dorsi muscle, 2.99 ± 0.50; liver, 2.45 ± 0.60; kidney, 3.81 ± 0.97; lung, 1.95 ± 0.14; and colon, 1.36 ± 0.29. Protein synthesis in the other mucin-producing tissues (i.e., stomach, proximal jejunum, midjejunum, and ileum) did not change with decreasing threonine concentrations, but mucin synthesis in the ileum and colon decreased over threonine concentrations <4.54 ± 1.50 and <3.20 ± 4.70 g/100 g total AAs, respectively. CONCLUSIONS: The results of this study illustrate that dietary threonine is preferentially used for protein synthesis in gastrointestinal tissues in piglets. If dietary threonine intake is deficient, then muscle growth and the functions of other tissues are likely compromised at the expense of maintenance of the mucus layer in mucin-producing tissues.

Dietary Methyl Donors Contribute to Whole-Body Protein Turnover and Protein Synthesis in Skeletal Muscle and the Jejunum in Neonatal Piglets.

BACKGROUND: The neonatal methionine requirement must consider not only the high demand for rapid tissue protein expansion but also the demands as the precursor for a suite of critical transmethylation reactions. However, methionine metabolism is inherently complex because upon transferring its methyl group during transmethylation, methionine can be reformed by the dietary methyl donors choline (via betaine) and folate. OBJECTIVE: We sought to determine whether dietary methyl donors contribute to methionine availability for protein synthesis in neonatal piglets. METHODS: Yucatan miniature piglets aged 4-8 d were fed a diet that provided 38 µg folate/(kg·d), 60 mg choline/(kg·d), and 238 mg betaine/(kg·d) [methyl-sufficient (MS); n = 8] or a diet devoid of these methyl precursors [methyl-deficient (MD); n = 8]. After 5 d, dietary methionine was reduced from 0.30 to 0.20 g/(kg·d) in both groups. On day 6, piglets received a constant [1-13C]phenylalanine infusion to measure whole-body protein kinetics, and on day 8 they received a constant [3H-methyl]methionine infusion to measure tissue-specific protein synthesis in skeletal muscle, the liver, and the jejunum. RESULTS: Whole-body phenylalanine flux, protein synthesis, and protein breakdown were 13%, 12%, and 22% lower, respectively, in the MD group than in the MS group (P < 0.05). Reduced whole-body protein synthesis in the MD piglets was attributed to 50% lower protein synthesis in skeletal muscle and the jejunum than in the MS piglets (P < 0.05). Furthermore, methionine availability in skeletal muscle was halved in piglets fed the MD diet (P < 0.05), and the specific radioactivity of methionine was doubled in the jejunum of MD piglets (P < 0.05), suggesting lower intestinal remethylation. Liver protein synthesis did not significantly differ between the groups, but secreted proteins were not measured. CONCLUSIONS: Dietary methyl donors can affect whole-body and tissue-specific protein synthesis in neonatal piglets and should be considered when determining the methionine requirement.

Dietary methyl donors affect in vivo methionine partitioning between transmethylation and protein synthesis in the neonatal piglet.

Methionine metabolism is critical during development with significant requirements for protein synthesis and transmethylation reactions. However, separate requirements of methionine for protein synthesis and transmethylation are difficult to define because after transmethylation, demethylated methionine is either irreversibly oxidized to cysteine during transsulfuration, or methionine is regenerated by the dietary methyl donors, choline (via betaine) or folate during remethylation. We hypothesized that remethylation contributes significantly to methionine availability and affects partitioning between protein and transmethylation. 4-8-day-old neonatal piglets were fed a diet devoid (MD-) (n = 8) or replete (MS+) (n = 8) of folate, choline and betaine to limit remethylation. After 5 days, dietary methionine was reduced to 80 % of requirement in both groups of piglets to ensure methionine availability was limited. On day 7, an intragastric infusion of [13C1]methionine and [2H3-methyl]methionine was administered to measure methionine cycle flux. In MD- piglets, in vivo remethylation was 60 % lower despite 23-fold greater conversion of choline to betaine (P < 0.05) and transmethylation was 56 % lower (P < 0.05), suggesting dietary methyl donors spared 425 µmol methyl/day for transmethylation. The priority of protein synthesis versus transmethylation was clear during MD- feeding (P < 0.05), as an additional 6 % of methionine flux was for protein synthesis in those piglets (P < 0.05). However, whole body transsulfuration was unaffected in vivo despite reduced in vitro cystathionine-ß-synthase capacity in MD- piglets (P < 0.05). Our data show that remethylation contributes significantly to methionine availability and that transmethylation is sacrificed to maintain protein synthesis when methionine is limiting in neonates, which should be considered when determining the methionine requirement.

The Trichoptera barcode initiative: a strategy for generating a species-level Tree of Life.

DNA barcoding was intended as a means to provide species-level identifications through associating DNA sequences from unknown specimens to those from curated reference specimens. Although barcodes were not designed for phylogenetics, they can be beneficial to the completion of the Tree of Life. The barcode database for Trichoptera is relatively comprehensive, with data from every family, approximately two-thirds of the genera, and one-third of the described species. Most Trichoptera, as with most of life's species, have never been subjected to any formal phylogenetic analysis. Here, we present a phylogeny with over 16 000 unique haplotypes as a working hypothesis that can be updated as our estimates improve. We suggest a strategy of implementing constrained tree searches, which allow larger datasets to dictate the backbone phylogeny, while the barcode data fill out the tips of the tree. We also discuss how this phylogeny could be used to focus taxonomic attention on ambiguous species boundaries and hidden biodiversity. We suggest that systematists continue to differentiate between 'Barcode Index Numbers' (BINs) and 'species' that have been formally described. Each has utility, but they are not synonyms. We highlight examples of integrative taxonomy, using both barcodes and morphology for species description.This article is part of the themed issue 'From DNA barcodes to biomes'.

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet.

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4-8 days of age were fed a diet deficient (MD-) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [(3)H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD- feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD- feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD- feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.