Evaluation of laboratory and on-farm tests to estimate colostrum quality for dairy cows.

The objectives of this study were to evaluate different analytical methods to determine colostrum quality in dairy cattle, including one laboratory-based method (ELISA) and 4 on-farm tests. We hypothesized that the colostral IgG concentration using different analytical methods, such as ELISA (mg/mL), digital Brix refractometer (% Brix), colostrometer (specific gravity and mg/mL), an outflow funnel (seconds), and a lateral flow assay (mg/mL), were highly correlated with the reference method, radial immunodiffusion (RID; mg/mL) and would generate comparable results. Colostrum samples were collected from 209 Holstein Friesian cows on 2 commercial dairy farms in Germany. Colostrum weight and colostrum temperature were measured. Test characteristics, such as optimum thresholds, sensitivity, specificity, and area under the curve (AUC) were determined using a receiver operating characteristic curve analyses for each test. Out of 209 colostrum samples assessed by RID, 186 (89%) samples had high quality (≥50 mg IgG/mL), while 23 colostrum samples (11%) showed poor quality with IgG concentrations less than 50 mg/mL. The mean IgG concentration (±SD) was 101.3 ± 45.9 mg/mL and the range was 6.0 to 244.3 mg/mL. The Pearson correlation coefficient (r) between RID and ELISA was r = 0.78. In comparison to RID, Pearson correlation coefficients for the on-farm tests were: r = 0.79 (digital Brix refractometry), r = 0.58 (colostrometer: specific gravity), r = 0.61 (colostrometer: temperature corrected), r = 0.26 (outflow funnel) and r = 0.43 (lateral flow assay), respectively. The optimal threshold to identify high-quality colostrum using ELISA was 50.8 mg/mL with sensitivity 91.3%, specificity 92.3%, and AUC of 0.94. For the on-farm tests sensitivity ranged from 95.7% (Brix refractometry) to 60.9% (lateral flow assay). Specificity ranged from 88.6% (lateral flow assay) to 75.9% (colostrometer: temperature corrected). The AUC ranged from 0.93 (Brix refractometry) to 0.73 (outflow funnel). Based on the AUC, ELISA (0.94) and Brix refractometry (0.93) can be considered highly accurate. In conclusion, the ELISA is accurate to assess colostrum quality. Regarding the on-farm tests only the digital Brix refractometer and the colostrometer were adequate to determine colostrum quality.

Hot topic: Accuracy of refractometry as an indirect method to measure failed transfer of passive immunity in dairy calves fed colostrum replacer and maternal colostrum.

Serum total protein (STP) refractometry is a widely used indicator of failed transfer of passive immunity (FTPI), defined as serum IgG concentrations of <10 mg/mL or STP levels <5.2 g/dL measured at 24 h of life. However, recent reports have demonstrated that refractometry could be inaccurate at estimating serum IgG concentrations and FTPI when calves are fed colostrum replacer (CR). The objective of this study was to evaluate the accuracy of STP measurements to estimate FTPI in calves fed CR compared with calves fed maternal colostrum. Blood was collected from dairy calves fed maternal colostrum (n = 927) or colostrum-derived CR (n = 1,258) and analyzed for STP and serum IgG. Serum total protein was measured with a digital refractometer, whereas radial immunodiffusion was used to determine IgG concentrations. Calves fed maternal colostrum had a mean STP of 5.80 ± 0.72 (standard deviation) g/dL and a mean IgG concentration of 22.81 ± 10.14 mg/mL, respectively, whereas calves fed CR had a mean STP and IgG concentration of 5.14 ± 0.50 g/dL and 12.78 ± 4.60 mg/mL, respectively. Rates of FTPI for calves fed maternal colostrum or CR were 4.2% and 27.26%, respectively. Calves were considered to have FTPI if their IgG postcolostrum feeding was <10 mg/mL. Logistic and linear regression analyses were performed to determine cutoff points and existent relationships between STP and IgG. Serum total protein and IgG for calves fed maternal colostrum were highly correlated. In contrast, STP and IgG for calves fed CR were lowly correlated. A receiver operator characteristic curve analysis demonstrated that an STP cutoff point that could predict FTPI when calves are fed CR would be 4.9 g/dL (sensitivity = 0.68; specificity = 0.75). This study suggests that current cutoff points used for STP inflates the number of calves estimated to have FTPI when they are fed CR.

Validation of Brix refractometers and a hydrometer for measuring the quality of caprine colostrum.

On-farm assessment of caprine colostrum quality is important for goat farmers; the ability to quickly recognize whether colostrum is suitable to feed to kids helps achieve successful passive transfer of immunity. The study compared the use of optical and digital Brix refractometers and a hydrometer against the international gold standard radial immunodiffusion (RID), using both fresh and frozen samples. A locally available ELISA methodology was included for comparison. A total of 300 samples were collected from 2 farms (farm 1: n = 157, collected by research staff within 24 h of parturition; farm 2: n = 143, collected by the farmer within 12 h of parturition). Farm 1 provided doe age for a subset of samples (n = 86). Samples were tested fresh and then frozen for shipment and repeated testing. Specific gravity was measured using a hydrometer in a subset of samples (n = 22) from farm 2. Because no gold standard thresholds are currently available for caprine colostrum, RID-derived values of 30, 40, and 50 g/L IgG were used as potential "good quality" thresholds. Pearson (ρ) and Lin's concordance correlation coefficients (CCC) were calculated for comparison of methods. Optimum thresholds were established maximizing the Youden index and minimizing the "distance closest to the top left corner" of the receiver operator characteristic curves. Brix values were correlated with RID (optical Brix, fresh: ρ = 0.73; digital Brix, fresh: ρ = 0.71; digital Brix, frozen: ρ = 0.76) and with each other (range: ρ = 0.93 to 0.99; CCC = 0.91 to 0.99). Specific gravity measured by the hydrometer yielded a strong relationship with RID (ρ = 0.83) and with Brix values (range: ρ = 0.88 to 0.90). The ELISA method was not correlated with Brix methods (range: ρ = 0.02 to 0.09) or RID (ρ = 0.20). Depending on the colostrum IgG threshold, the hydrometer yielded high Youden indices (range: 0.78 to 0.93) and low distance closest to the top left corner criteria (0 to 0.05) at a threshold of 1.047 specific gravity. For all RID IgG thresholds, the best Brix threshold (regardless of type or whether the sample was fresh or frozen) was 18 or 19%, with the highest Youden indices (range: 0.47 to 0.61) and lowest distance to the top left corner criteria (range: 0.09 to 0.16); however, we recommend 19%, because this reduces the potential of feeding poor-quality colostrum. The ELISA method was the poorest predictor of colostrum concentration. Age was not found to affect colostrum quality; however, the sample size of this subset was small. Hydrometers are inexpensive and easy to use, whereas Brix methods use only a small amount of colostrum; we suggest that either method could be used on-farm.

Evaluation of different analytical methods to assess failure of passive transfer in neonatal calves.

The objective of this study was to evaluate different analytical methods of assessing failure of passive transfer (FPT) in neonatal calves. We hypothesized that 3 different media (i.e., centrifuged serum, centrifuged plasma, filtered plasma) and different analytical methods [i.e., ELISA, capillary electrophoresis (CE), Brix refractometer, and handheld optical refractometer] would be highly correlated with the gold standard radial immunodiffusion (RID) and would generate comparable results. Serum and plasma blood samples were collected from Holstein Friesian calves (n = 216) aged 1 to 7 d, from 2 commercial dairy herds in northeast Germany. The RID analysis showed that 59 of 216 calves (27%) had serum IgG concentrations of <10 mg/mL and 157 calves (73%) had serum concentrations of ≥10 mg/mL. The mean IgG concentration (± standard deviation) was 17.1 ± 9.8 mg/mL, and the range was 0.8 to 47.8 mg/mL. In serum, the correlation between RID and CE was r = 0.97, and between RID and ELISA was r = 0.90; CE and ELISA were also highly correlated (r = 0.89). Both refractometry methods were highly correlated with RID using centrifuged serum, centrifuged plasma, or filtered plasma (Brix refractometer: r = 0.84, 0.80, and 0.78, respectively; handheld optical refractometer: r = 0.83, 0.81, and 0.80, respectively). We determined test characteristics (optimum thresholds, sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve) for CE, ELISA, and the handheld optical and digital refractometers using receiver operating characteristic curve analyses with RID as the reference value. Optimal thresholds for assessing FPT using plasma were higher than for serum, regardless of the method of plasma harvesting. The 4 different devices had comparable areas under the curve, irrespective of the medium used. All analytical methods can be used to assess FPT.

Preweaned heifer management on US dairy operations: Part II. Factors associated with colostrum quality and passive transfer status of dairy heifer calves.

Passive transfer of immunity is essential for the short- and long-term health of dairy calves. The objective of this study was to evaluate factors associated with colostrum quality and passive transfer status of US heifer calves. This study included 104 operations in 13 states that participated in the calf component of the National Animal Health Monitoring System's Dairy 2014 study. This 18-mo longitudinal study included 1,972 Holstein heifer calves from birth to weaning. Multivariable mixed linear regression models were selected using backward elimination model selection after univariate screening to determine which factors were associated with colostrum IgG and serum IgG concentrations. The mean colostrum IgG concentration was 74.4 g/L with 77.4% of colostrum samples having IgG concentrations >50 g/L. The final model for colostrum IgG included colostrum source and a categorized temperature-humidity index value (cTHI) for the month before calving. Mean colostrum IgG concentrations were highest for dams in third and higher lactations (84.7 g/L) and lowest for commercial colostrum replacers (40.3 g/L). Colostrum IgG concentrations were highest for cTHI ≥70 (72.6 g/L) and lowest for cTHI <40 (64.2 g/L). The mean serum IgG concentration was 21.6 g/L, with 73.3% of calves having serum IgG concentrations >15 g/L. The final model for serum IgG concentration included region, heat treatment of colostrum, colostrum source, timing to first feeding, volume of colostrum fed in the first 24 h, age of the calf at blood sampling, and colostrum IgG concentration. Mean serum IgG concentrations were highest for calves that received colostrum from first-lactation dams (25.7 g/L) and lowest for calves fed commercial colostrum replacer (16.6 g/L). Serum IgG concentrations were higher for calves fed heat-treated colostrum (24.4 g/L) than for calves fed untreated colostrum (20.5 g/L). Serum IgG concentration was positively associated with the volume of colostrum fed in the first 24 h and colostrum IgG concentration, and negatively associated with the number of hours from birth to colostrum feeding and age (days) at blood collection. Dairy producers should be encouraged to measure the quality of colostrum before administering it to calves and to measure serum IgG or a proxy such as serum total protein or Brix to evaluate passive immunity and colostrum management programs.

Evidence for rapid evolutionary change in an invasive plant in response to biological control.

We present evidence that populations of an invasive plant species that have become re-associated with a specialist herbivore in the exotic range through biological control have rapidly evolved increased antiherbivore defences compared to populations not exposed to biocontrol. We grew half-sib families of the invasive plant Lythrum salicaria sourced from 17 populations near Ottawa, Canada, that differed in their history of exposure to a biocontrol agent, the specialist beetle Neogalerucella calmariensis. In a glasshouse experiment, we manipulated larval and adult herbivory to examine whether a population's history of biocontrol influenced plant defence and growth. Plants sourced from populations with a history of biocontrol suffered lower defoliation than naïve, previously unexposed populations, strongly suggesting they had evolved higher resistance. Plants from biocontrol-exposed populations were also larger and produced more branches in response to herbivory, regrew faster even in the absence of herbivory and were better at compensating for the impacts of herbivory on growth (i.e. they exhibited increased tolerance). Furthermore, resistance and tolerance were positively correlated among genotypes with a history of biocontrol but not among naïve genotypes. Our findings suggest that biocontrol can rapidly select for increased defences in an invasive plant and may favour a mixed defence strategy of resistance and tolerance without an obvious cost to plant vigour. Although rarely studied, such evolutionary responses in the target species have important implications for the long-term efficacy of biocontrol programmes.

Evaluating different methods of serum collection to detect failed transfer of passive immunity in newborn calves via refractometry.

The objective of the study was to compare 4 different methods of serum collection to assess failed transfer of passive immunity (FTPI) in dairy calves. We hypothesized that centrifuged serum, filtered serum and clotted serum at room temperature, and clotted serum at refrigerator temperature measured with Brix refractometry would highly correlate with IgG concentration assessed by radial immunodiffusion (RID; gold standard) in centrifuged serum. Blood samples were collected from 321 newborn dairy calves. In centrifuged serum (r = 0.88), serum clotted at room temperature (20.2°C ± 6.47; r = 0.86), serum clotted at refrigerator temperature (7.6°C ± 0.91; r = 0.87), and filtered serum (r = 0.70), total solids (TS) in % Brix, and IgG concentrations measured with RID were highly correlated. Regarding the refractometry results among the different serum types, the TS results of serum clotted at room temperature, clotted at refrigerator temperature, and filtered serum showed high correlation coefficients compared with the TS results of centrifuged serum (r = 0.99, r = 0.98, and r = 0.89), respectively. The test characteristics of clotted serum were as accurate as centrifuged serum and generate comparable results. Filtered serum was slightly less accurate. All serum types are valid methods to detect an FTPI in dairy calves, if the specific Brix thresholds for each serum type are considered. Nevertheless, serum clotted at refrigerator temperature should not be the preferred method to avoid the risk of hemolysis.

Colostrum insulin supplementation does not influence immunoglobulin G absorption in neonatal Holstein bulls.

Since insulin has been demonstrated to suppress IgG absorption in other neonatal species, we had the objective to delineate how colostral insulin concentrations affect IgG absorption in neonatal bovines. We enrolled Holstein bull calves (n = 48; body weight = 46.3 ± 0.84 kg) at birth and randomized them by birth order to receive (1) colostrum that contained basal insulin concentrations (12.9 µg/L; n = 16), or colostrum that had been supplemented with an exogenous insulin to increase the insulin concentration to either (2) 5 times (70.0 µg/L; n = 16) or (3) 10 times (149.7 µg/L; n = 16) that of the basal colostrum. Gross colostrum composition (crude fat: 4.1 ± 0.06%; crude protein: 11.7 ± 0.05%; lactose: 1.9 ± 0.01%; IgG: 63.9 ± 1.19 g/L) was similar between treatments and calves were fed (7% body weight, 3.1 ± 0.06 L) their treatments at 2, 14, and 26 h postnatal. Serum was collected at 0, 30, 60, 90, 120, 180, 240, 360, 480, and 600 min postprandial respective to the first and second colostrum feeding and analyzed for IgG concentration. The incremental area under the curve (I-AUC) and apparent efficiency of absorption (AEA) were calculated for the 10-h periods following the first and second colostrum meal. Serum IgG concentrations over time, I-AUC, and AEA were statistically analyzed as a complete randomized design. Colostrum insulin concentration did not affect serum IgG concentrations or the I-AUC or AEA after calves were fed colostrum at 2 and 14 h postnatal. High colostral insulin content is not detrimental or promotive to IgG absorption in neonatal Holstein bulls.

Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native "wildtype" DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach. Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells. Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

No evidence that rapid adaptation impedes biological control of an invasive plant.

Biological control is a popular tool for invasive species management, but its success in nature is difficult to predict. One risk is that invasive plants, which may have adapted to lower herbivore pressure in the introduced range, could rapidly evolve defences upon re-association with their biocontrol agent(s). Previous studies have demonstrated that populations of the invasive plant purple loosestrife (Lythrum salicaria) exposed to biocontrol exhibit traits consistent with the rapid evolution of defence. However, to date, no one has tested this hypothesis under field-natural levels of herbivory. Using seed from 17 populations of purple loosestrife growing in eastern Canada, that varied in their history of exposure to their biocontrol agent, the leaf beetle Neogalerucella spp., we transplanted 1,088 seedlings from 136 maternal families into a common garden under ambient herbivory. Over the following three and half years, we assessed plant performance in the face of biocontrol by measuring early-season plant size, defoliation, flowering, and season-end biomass. We discovered that a population history with biocontrol explained little variation in herbivory or plant performance, suggesting that adaptation is not hindering biocontrol effectiveness. Instead, plant size, subsequent defoliation, and spatio-temporal variables were the main predictors of plant growth and flowering during the study. The high individual variability we observed in plant performance underscores that flexible strategies of allocation and phenology are important contributors to the persistence of invasive plants. Our findings suggest that plant adaptation to biocontrol is unlikely to be a strong impediment to biological control in this species, however, the high survival and variable defoliation of plants in our study also indicate that biocontrol alone is unlikely to result in significant population decline. We recommend that the application of multiple forms of control simultaneously (e.g. thinning plus biocontrol) could help to prevent the existence of refuges of large, reproductive individuals.

Seasonal differences in the efficacy of pour-on formulations of triclabendazole and ivermectin or abamectin against late immature liver fluke (Fasciola hepatica) in cattle.

Three controlled studies were conducted to determine the efficacy against late immature (6 weeks) Fasciola hepatica of two currently available fasciolicides (Genesis Ultra and Coopers Sovereign) which are applied externally to cattle. Efficacy of the two products was assessed when application was made under winter, spring and summer conditions. Efficacies for winter, spring and summer respectively, based on arithmetic mean total fluke counts, were 78.9%, 91.7% and 99.6% for Coopers Sovereign and 73.4%, 89.7% and 99.6% for Genesis Ultra. Seasonal differences with treatment efficacy were indicated. The studies also confirmed previous observations that liver fluke egg counts overestimate the efficacy of fasciolicides and that total fluke counts is the most reliable method for assessing efficacy of such products.

Mucoepidermoid carcinoma of upper aerodigestive tract: clinicopathologic study of 78 cases with immunohistochemical analysis of Dicer expression.

MicroRNAs (miR) are small noncoding RNAs that are predicted to regulate up to 30% of protein-encoding genes. miR maturation requires functional microRNA machinery, including the Dicer protein. We review our experience with mucoepidermoid carcinoma (MEC) and characterize the prognostic value of Dicer expression. Expression of Dicer was assessed in 78 MEC by immunohistochemistry. Dicer expression was scored semiquantitatively and relative to the internal controls: large excretory/striated ducts or basal/parabasal layers of normal squamous epithelium (mucosa). Dicer scores were then correlated with clinical and pathologic parameters. Dicer over- and/or under-expression were more commonly seen in high-grade MEC (83%) than in low/intermediate grade MEC (35%; p=0.002) and in stage III/IV MEC (80%) than in stage I/II MEC (41%; p=0.04). Abnormal Dicer expression correlates with high-grade and advanced stage, acting as a univariate predictor of poor disease-specific survival (DSS) in MEC. Age and stage were independent predictors of poor DSS on multivariate analysis. Abnormal immunoexpression of Dicer in aggressive MEC suggests a role for miR and miR machinery in tumor progression.

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

Insertion of a telomere repeat sequence into a mammalian gene causes chromosome instability.

Telomere repeat sequences cap the ends of eucaryotic chromosomes and help stabilize them. At interstitial sites, however, they may destabilize chromosomes, as suggested by cytogenetic studies in mammalian cells that correlate interstitial telomere sequence with sites of spontaneous and radiation-induced chromosome rearrangements. In no instance is the length, purity, or orientation of the telomere repeats at these potentially destabilizing interstitial sites known. To determine the effects of a defined interstitial telomere sequence on chromosome instability, as well as other aspects of DNA metabolism, we deposited 800 bp of the functional vertebrate telomere repeat, TTAGGG, in two orientations in the second intron of the adenosine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. In one orientation, the deposited telomere sequence did not interfere with expression of the APRT gene, whereas in the other it reduced mRNA levels slightly. The telomere sequence did not induce chromosome truncation and the seeding of a new telomere at a frequency above the limits of detection. Similarly, the telomere sequence did not alter the rate or distribution of homologous recombination events. The interstitial telomere repeat sequence in both orientations, however, dramatically increased gene rearrangements some 30-fold. Analysis of individual rearrangements confirmed the involvement of the telomere sequence. These studies define the telomere repeat sequence as a destabilizing element in the interior of chromosomes in mammalian cells.

Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells.

Expansion of CTG triplet repeats in the 3' untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG)(17), (CTG)(98), and (CTG)(183) repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG)(98) and (CTG)(183), 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG)(98) and (CTG)(183) cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG)(17) repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.

Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks.

Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT's ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT's activity at DNA ends in vivo.

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells.

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Acute chest pain in the emergency room. Identification and examination of low-risk patients.

Clinical and laboratory data from 596 patients who came to an emergency room complaining of chest pain indicated that no single variable could identify low-risk patients as well as a normal ECG. A combination of three variables--sharp or stabbing pain, no history of angina or myocardial infarction, and pain with pleuritic or positional components or pain that was reproduced by palpation of the chest wall--defined a very-low-risk group in which ECGs did not add accuracy to the evaluation and were potentially misleading; however, only 48 patients (8%) fell into this category. Standard cardiac enzyme levels were of almost no use as an emergency room indicator of myocardial infarction. These findings emphasize the difficulty of identifying patients at low risk for myocardial infarction or unstable angina in the emergency room without consideration of many factors from the history, the physical examination, and the ECG.

Design and synthesis of metabolically stable atrial natriuretic factor analogs. Amino- and carboxy-terminal stabilization.

Two analogs of rat atrial natriuretic factor, rANF7-28-NH2 and [Mpr7,Ala20,D-Arg27]rANF7-27-NH2, were prepared by the solid-phase method. These peptides had 2-fold and 7-fold less affinity, respectively, than rANF1-28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5-fold less potent than rANF1-28 in relaxing serotonin-contracted rabbit aortic rings. rANF7-28-NH2 was rapidly degraded by rat kidney homogenates but [Mpr7,Ala20,D-Arg27]rANF7-27-NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.