Temperature-Dependent Biomarkers of Estrogenic Exposure in a Piscivore Freshwater Fish.

The biological effects of endocrine-active compounds and increasing water temperatures as a result of climate change have been studied extensively and independently, but there is a dearth of research to examine the combined effect of these factors on exposed organisms. Recent data suggest that estrogenic exposure and rising ambient temperatures independently impact predator-prey relationships. However, establishing these connections in natural settings is complex. These obstacles can be circumvented if biomarkers of estrogenic exposure in resident fish can predict changes in predator-prey relationships. To test the effects of estrone and temperature, the piscivore bluegill sunfish (Lepomis macrochirus) was exposed for 30 days to estrone at concentrations (90 ± 17.6 ng/L [mean ± standard deviation] and 414 ± 146 ng/L) previously shown to reduce prey-capture success. Exposures were conducted at four temperatures (15 °C, 18 °C, 21 °C, 24 °C) to simulate breeding season ambient temperatures across the natural range of this species. A suite of morphological and physiological biomarkers previously linked to estrogenic exposures were examined. Biomarkers of estrone exposure were more commonly and severely impacted in male fish than in female fish. Notably, the gonadosomatic index was lower and gonads were less mature in exposed males. Additionally, temperature modulated the effects of estrone similarly in males and females with fish exposed at higher temperatures typically exhibiting a decreased morphological index. This study provides evidence that alterations in hepatic function and gonadal function may cause shifts in metabolism and energy allocation that may lead to declining prey capture performance.

Thermal modulation of anthropogenic estrogen exposure on a freshwater fish at two life stages.

Human-mediated environmental change can induce changes in the expression of complex behaviors within individuals and alter the outcomes of interactions between individuals. Although the independent effects of numerous stressors on aquatic biota are well documented (e.g., exposure to environmental contaminants), fewer studies have examined how natural variation in the ambient environment modulates these effects. In this study, we exposed reproductively mature and larval fathead minnows (Pimephales promelas) to three environmentally relevant concentrations (14, 22, and 65ng/L) of a common environmental estrogen, estrone (E1), at four water temperatures (15, 18, 21, and 24°C) reflecting natural spring and summer variation. We then conducted a series of behavioral experiments to assess the independent and interactive effects of temperature and estrogen exposure on intra- and interspecific interactions in three contexts with important fitness consequences; reproduction, foraging, and predator evasion. Our data demonstrated significant independent effects of temperature and/or estrogen exposure on the physiology, survival, and behavior of larval and adult fish. We also found evidence suggesting that thermal regime can modulate the effects of exposure on larval survival and predator-prey interactions, even within a relatively narrow range of seasonally fluctuating temperatures. These findings improve our understanding of the outcomes of interactions between anthropogenic stressors and natural abiotic environmental factors, and suggest that such interactions can have ecological and evolutionary implications for freshwater populations and communities.

Inhalation anesthesia induced by isoflurane alters penicillin disposition in swine tissues.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24 h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28 h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8 h postdose, and a second set of four similarly treated control pigs were sacrificed 28 h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

Triticum monococcum lines with distinct metabolic phenotypes and phloem-based partial resistance to the bird cherry-oat aphid Rhopalosiphum padi.

Crop protection is an integral part of establishing food security, by protecting the yield potential of crops. Cereal aphids cause yield losses by direct damage and transmission of viruses. Some wild relatives of wheat show resistance to aphids but the mechanisms remain unresolved. In order to elucidate the location of the partial resistance to the bird cherry-oat aphid, Rhopalosiphum padi, in diploid wheat lines of Triticum monococcum, we conducted aphid performance studies using developmental bioassays and electrical penetration graphs, as well as metabolic profiling of partially resistant and susceptible lines. This demonstrated that the partial resistance is related to a delayed effect on the reproduction and development of R. padi. The observed partial resistance is phloem based and is shown by an increase in number of probes before the first phloem ingestion, a higher number and duration of salivation events without subsequent phloem feeding and a shorter time spent phloem feeding on plants with reduced susceptibility. Clear metabolic phenotypes separate partially resistant and susceptible lines, with the former having lower levels of the majority of primary metabolites, including total carbohydrates. A number of compounds were identified as being at different levels in the susceptible and partially resistant lines, with asparagine, octopamine and glycine betaine elevated in less susceptible lines without aphid infestation. In addition, two of those, asparagine and octopamine, as well as threonine, glutamine, succinate, trehalose, glycerol, guanosine and choline increased in response to infestation, accumulating in plant tissue localised close to aphid feeding after 24 h. There was no clear evidence of systemic plant response to aphid infestation.

Echocardiographic caudal vena cava measurements in healthy cats and in cats with congestive heart failure and non-cardiac causes of cavitary effusions.

BACKGROUND: Echocardiographic indices of the inferior vena cava have been associated with elevated right atrial pressures in humans. HYPOTHESIS/OBJECTIVES: Describe caudal vena caval (CVC) sonographic dimensions in healthy cats compared to cats with cardiogenic cavitary effusion (CCE), cardiogenic pulmonary edema (CPE), or non-cardiac causes of cavitary effusion (NCE). ANIMALS: 30 healthy control cats and 52 client-owned cats with CCE, CPE, or NCE examined at two university hospitals. METHODS: Sagittal 2-dimensional (2D) and M-mode CVC dimensions were acquired from the subxiphoid view. Caudal vena cava collapsibility index (CVC-CI) was calculated. Variables were compared between study groups using Kruskal-Wallis and Dunn's Bonferroni testing. Receiver operating characteristic curves were used to assess sensitivity and specificity for diagnostic categories. RESULTS: Healthy cats had sagittal 2D and M-mode (median, interquartile range) CVC maximal dimensions of 2.4 mm (1.3-4.0) and 3.4 mm (1.5-4.9) and CVC-CI of 52% (45.2-61.8) and 55% (47.8-61.3), respectively. The CVC maximal dimensions in healthy controls were smaller than in cats with cavitary effusions or pulmonary edema (all P<0.05). CVC-CI was different between CCE and NCE (P<0.0001) with cutoffs of CVC-CI ≤38% (2D) or ≤29% (M-mode) being 90.5% and 85.7% sensitive, and 94.4% and 100% specific for diagnosis of CCE, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Caudal vena cava measurements are larger in cats with cavitary effusions and cats with CPE than healthy cats. In cats with cavitary effusion, decreased CVC-CI, ≤38% (2D) or ≤29% (M-mode), was helpful in distinguishing between cardiogenic and noncardiogenic etiology.

Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification.

The discovery of biomarkers in milk indicative of local inflammation or disease in the bovine mammary gland has been hindered by the extreme biological complexity of milk, the dynamic range of proteins in the matrix that renders the identification of low-abundance proteins difficult, and the challenges associated with quantifying changes during disease in the abundance of proteins for which no antibody exists. The objectives of the current study were to characterize the temporal expression of milk proteins following Escherichia coli challenge and to evaluate change in relative abundance of identified proteins using a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) label-free semiquantitative approach. Liquid chromatography-MS/MS conducted on whey from milk samples collected just before infusion with E. coli and at 12, 18, 24, 36, 48, and 60h following infection resulted in the identification of the high- to medium-abundance proteins alpha(S1)-, alpha(S2)- beta-, and kappa-caseins and the whey proteins serum albumin, beta-lactoglobulin, and alpha-lactalbumin. Additionally, a select number of lower abundance markers of inflammation were also identified, including lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein, and cyclic dodecapeptide-1. Normalized peptide counts for each protein identified were used to evaluate temporal changes in milk proteins following infection. For comparison with relative protein abundance determined using proteomic-based methods, changes in serum albumin, lactoferrin, and transferrin in milk during disease were also measured using ELISA. Label-free, proteomic-based quantification revealed relative changes in milk proteins that corresponded to expression profiles generated by ELISA. The results indicate that label-free LC-MS/MS methods are a viable means of tracking changes in relative protein abundance in milk during disease. Despite the identification of primarily abundant milk proteins, the results indicate that, with further refinement, LC-MS/MS could be used to evaluate temporal changes in proteins related to host response for which no antibody or ELISA currently exists.

Temperature and Estrogen Alter Predator-Prey Interactions between Fish Species.

A variety of environmental estrogens are commonly detected in human-impacted waterways. Although much is known about the effects of these environmental estrogens on the reproductive physiology and behavior of individuals within species, comparatively less is known about how these compounds alter the outcomes of interactions between species. Furthermore, few studies have considered how the effects of contaminants are modulated by natural variation in abiotic factors, such as temperature. To help fill this knowledge gap, we conducted a factorial experiment to examine the independent and combined effects of estrone (E1) and temperature on the outcome of predator-prey interactions between two common North American freshwater fishes, fathead minnows (Pimephales promelas) and bluegill sunfish (Lepomis macrochirus). Larval fathead minnows and adult sunfish were exposed to either a low (mean±standard deviation, 90.1 ± 18 ng/L; n = 16) or high (414 ± 147 ng/L; n = 15) concentration of E1 or to a solvent control for 30 days at one of four natural seasonal temperatures (15°C, 18°C, 21°C, and 24°C) before predation trials were performed. Exposure to E1 was associated with a significant increase in larval predation mortality that was independent of temperature. Across all temperature treatments, approximately 74% of control minnows survived; this survivorship significantly exceeded that of minnows exposed to either concentration of E1 (49% and 53% for minnows exposed to the low and high concentrations, respectively). However, exposure to E1 also impaired the prey-capture success of sunfish, partially mitigating predation pressure on exposed minnows. Overall prey-capture success by sunfish showed an inverted U-shaped distribution with temperature, with maximal prey consumption occurring at 21°C. This study illustrates the vulnerability of organismal interactions to estrogenic pollutants and highlights the need to include food web interactions in assessments of risk.

Do modern types of wheat have lower quality for human health?

Wheat is the major staple food in Western Europe and an important source of energy, protein, dietary fibre, minerals, B vitamins and phytochemicals. Plant breeders have been immensely successful in increasing yields to feed the growing global population. However, concerns have been expressed that the focus on increasing yield and processing quality has resulted in reduced contents of components that contribute to human health and increases in adverse reactions. We review the evidence for this, based largely on studies in our own laboratories of sets of wheats bred and grown between the 18th century and modern times. With the exception of decreased contents of mineral micronutrients, there is no clear evidence that intensive breeding has resulted in decreases in beneficial components or increases in proteins which trigger adverse responses. In fact, a recent study of historic and modern wheats from the UK showed increases in the contents of dietary fibre components and a decreased content of asparagine in white flour, indicating increased benefits for health.

Estimation of the iron bioavailability in green vegetables using an in vitro digestion/Caco-2 cell model.

It is estimated that over 30% of the global population is anaemic, half of which is due to iron deficiency. The bioavailability of iron from vegetables is low and variable, and influenced by food composition and matrix. We have therefore determined the relative bioavailability of iron in five types of green vegetable, spinach, broccoli, savoy cabbage, curly kale and green pepper, by measuring the ferritin response in a simulated digestion/Caco-2 cell model. Savoy cabbage gave the highest ferritin response and analysis of the digest showed that the iron was present in low molecular weight fractions which contained glucose, fructose, organic acids and amino acids. The addition of fructose 1,6-biphosphate to the Caco-2 cells increased iron uptake 2-fold. These results demonstrate that cabbage was the best source of bioavailable iron out of the vegetables studied and suggest that the formation of complexes with fructose derivatives contribute to increase the iron bioavailability.

Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis.

The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis on whey samples from a group of 8 cows before and 18 h after infection with Escherichia coli and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry post source decay. Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and beta-lactoglobulin, the low abundance proteins transthyretin, lactadherin, beta-2-microglobulin precursor, alpha-1-acid glycoprotein, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 h postinfection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18 h postinoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenecin 5 and 7, and the proteins beta-fibrinogen, alpha-2-HS-glycoprotein, S100-A12, and alpha-1-antiproteinase. Most notable was the detection of the acute phase protein alpha-1-acid glycoprotein in mastitic whey samples, a result not previously reported. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows after inoculation with E. coli could provide a rapid survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.

Temperature modulates estrone degradation and biological effects of exposure in fathead minnows.

Environmental pollutants, including estrogens, are widespread in aquatic environments frequently as a result of treated wastewater effluent discharged. Exposure to estrogens has been correlated with disruption of the normal physiological and reproductive function in aquatic organisms, which could impair the sustainability of exposed populations. However, assessing the effects of estrogen exposure on individuals is complicated by the fact that rates of chemical uptake and environmental degradation are temperature dependent. Because annual temperature regimes often coincide with critical periods of biological activity, temperature-dependent changes in estrogen degradation efficacy during wastewater treatment could modulate biological effects. We examined the interactions between ambient water temperature and degradation of estrone (E1) during wastewater treatment. In addition, we exposed mature fathead minnows (Pimephales promelas) to three environmentally relevant concentrations of E1 at four different water temperatures (15°C, 18°C, 21°C, and 24°C) to reflect natural seasonal variation. E1 degradation occurred with and without the support of robust nitrification at all temperatures; however, the onset of E1 degradation was delayed at cooler water temperatures. In addition, we observed significant interactive effects between temperature and E1 exposure. Female morphometric endpoints were more susceptible to temperature-modulating effects while physiological endpoints were more strongly affected in males. Collectively, the data demonstrate that natural seasonal fluctuations in temperature are sufficient to affect E1 degradation during wastewater treatment and induce sex-dependent physiological and anatomical changes in exposed fish.

Two Transduction Pathways Mediate Rapid Effects of Abscisic Acid in Commelina Guard Cells.

Commelina guard cells can be rapidly closed by abscisic acid (ABA), and it is thought that this signal is always transduced through increases in cytosolic calcium. However, when Commelina plants were grown at 10 to 17[deg]C, most guard cells failed to exhibit any ABA-induced increase in cytosolic calcium even though all of these cells closed. At growth temperatures of 25[deg]C or above, ABA-induced closure was always associated with an increase in cytosolic calcium. This suggests that there may be two transduction routes for ABA in guard cells; only one involves increases in cytosolic calcium. Activation of either pathway on its own appears to be sufficient to cause closure. Because the rates of ABA accumulation and transport in plants grown at different temperatures are likely to be different, we synthesized and microinjected caged ABA directly into guard cells. ABA was released internally by UV photolysis and subsequently caused stomatal closure. This result suggests a possible intracellular locale for the hypothesized ABA receptor.

Outcome and survival in canine sick sinus syndrome and sinus node dysfunction: 93 cases (2002-2014).

INTRODUCTION: To evaluate the clinical presentation, diagnosis, treatment, and outcomes of a group of dogs with sinoatrial node abnormalities. ANIMALS: Ninety-three client-owned dogs at a referral institution. MATERIALS AND METHODS: Medical records were reviewed for clinical history, diagnostic testing, and medical or permanent artificial pacemaker (PAP) treatment. Owners or veterinarians were contacted for long-term follow-up. RESULTS: Sixty-one dogs were symptomatic for their bradyarrhythmia and were diagnosed with sick sinus syndrome (SSS). Thirty-two dogs were asymptomatic for their bradyarrhythmia and were diagnosed with sinus node dysfunction (SND). Miniature Schnauzers, West Highland White terriers, Cocker spaniels, and female dogs were overrepresented. Medical management with positive chronotropic drugs successfully controlled syncope long-term in 54% of SSS dogs, and acted as a bridge to PAP in 20%. Positive atropine response predicted medical treatment success. Forty-six percent of SSS dogs eventually underwent PAP implantation. Median survival time was approximately 18 months in SND and SSS dogs regardless of treatment strategy. Congestive heart failure (CHF) associated with progressive valvular heart disease occurred commonly in all groups, particularly in dogs with bradycardia-tachycardia syndrome. CONCLUSIONS: Sinus node dysfunction and SSS represent a spectrum of sinoatrial node disease, which for some dogs may also involve a component of autonomic dysfunction. Dogs with SND do not require treatment. Dogs with SSS often require treatment to reduce the frequency of syncope; medical management is often useful, particularly in atropine responsive dogs. Prognosis of SSS with treatment is good, though development of CHF does not appear to be mitigated by treatment.

Multi-platform metabolomics analyses of a broad collection of fragrant and non-fragrant rice varieties reveals the high complexity of grain quality characteristics.

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

Alteration of morphogenesis by the v-myc oncogene in transplants of mammary gland.

To see if individual oncogenes can alter three-dimensional growth in vivo, we have inserted the v-myc oncogene into transplants of mouse mammary gland. Primary cultures of mammary epithelial cells were infected with helper-free retrovirus to insert v-myc oncogenes, and transplanted into cleared mammary fat pads (mammary glands from which the natural epithelium had been removed) of host mice. Uninfected transplants, and transplants infected with retrovirus constructs that carry no oncogene, grew to form an epithelial 'tree' resembling normal mammary gland, as expected. Transplants infected with retroviruses carrying v-myc oncogenes grew to form a characteristic, abnormal (hyperplastic) pattern in which the ducts were more densely packed than normal. Integration of the retrovirus in the transplants was demonstrated. The effect of the oncogene was local, not systemic, as some transplants showed adjacent areas of normal and hyperplastic growth. Thus v-myc can alter morphogenesis without growth becoming disorganized.

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.

The presence of type III collagen in the developing tooth.

Type I and type III collagens have been isolated from dental papilla and dental pulp of bovine tissues by enzymic digestion with pepsin and differential salt precipitation. Type III collagen was further purified by molecular sieve and ion-exchange chromatography. The relative proportions of type III and type I collagens were estimated by two different techniques, gel electrophoresis in the presence of mercaptoethanol, and direct estimation of the alpha1(III)CB3 peptide. Both techniques gave similar values. Type III collagen constituted 24% of papilla and 28% of pulp collagen by the first procedure, and 20% and 31% by the second. 18% of dental follicle collagen was type III.

Complication rates associated with transvenous pacemaker implantation in dogs with high-grade atrioventricular block performed during versus after normal business hours.

BACKGROUND: Transvenous pacemaker implantation in dogs is associated with a relatively high complication rate. At our institution, pacemaker implantation in dogs with high-grade atrioventricular block (HG-AVB) frequently is performed as an after-hours emergency. HYPOTHESIS: Among dogs with HG-AVB, the rate of major complications is higher when pacemakers are implanted after hours (AH) compared to during business hours (BH). ANIMALS: Client-owned dogs with HG-AVB that underwent transvenous pacemaker implantation between January 2002 and December 2012 at the North Carolina State University Veterinary Teaching Hospital. METHODS: Retrospective medical record review. Two-year follow-up was required for complications analysis. RESULTS: Major complications occurred in 14/79 dogs (18%) and included lead dislodgement, lead or generator infection, lead or generator migration, and pacing failure. Incidence of major complications was significantly higher AH (10/36, 28%) compared to BH (4/43, 9%; P = .041), and all infectious complications occurred AH. Median survival time for all dogs was 27 months and did not differ between AH and BH groups for either all-cause (P = .70) or cardiac (P = .40) mortality. AH dogs were younger than BH dogs (P = .010), but there were no other clinically relevant differences between BH and AH groups in terms of demographic, clinical, or procedural variables. CONCLUSIONS AND CLINICAL IMPORTANCE: At our institution, AH transvenous pacemaker placement is associated with a higher rate of major complications (especially infections) compared to BH placement. This difference may be because of a variety of human factor differences AH versus BH.

Absorption pharmacokinetics of atenolol in patients with the Marfan syndrome.

The negative inotropic and chronotropic effects of beta-blocker therapy have been reported to reduce morbidity and mortality in patients with Marfan syndrome; however, little is known about the pharmacokinetics of atenolol after oral administration of multiple doses to patients with the Marfan syndrome. We studied the pharmacokinetics of atenolol in 13 such patients aged 18.7 +/- 2.9 years who were receiving 1.78 +/- 0.58 mg/kg/day (70.1 +/- 20.3 mg/m2/day) of atenolol for 6 weeks or longer. Mean +/- SD percentage change in baseline heart rate after the administration of atenolol was -18.03 +/- 16.59% and mean +/- SD percentage change in exercise heart rate after atenolol was -33.22 +/- 14.75% (P < .01). Six to 8 atenolol serum concentrations were collected in each patient during a 12-hour dosing interval and were determined by high-performance liquid chromatography with ultraviolet detection. Serum atenolol concentrations at 0 (123 +/- 70 micrograms/L) and 12 (116 +/- 66 micrograms/L) hours were within 20% of each other and were thus assumed to be at steady-state. A one-compartment, steady-state pharmacokinetic model with first-order absorption and elimination was fitted to the concentration-time data for each patient using nonlinear regression. Maximal concentration was 343 +/- 120 micrograms/L, and the mean half-life was 4.72 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm microinjection twins: deoxyribonucleic acid fingerprinting.

The twins described are clearly nonidentical. The DNA fingerprints obtained show that they are the children of the putative mother and father, indicating the success of the microinjection procedure.