Long-Read Sequencing Genome Assembly of Ceratocystis fimbriata Enables Development of Molecular Diagnostics for Sweetpotato Black Rot.

Ceratocystis fimbriata is a destructive fungal pathogen of sweetpotato (Ipomoea batatas) that leads to losses at all stages of sweetpotato production. Accurate detection of C. fimbriata would allow for more efficient deployment of management tactics in sweetpotato production. To develop a diagnostic assay, a hybrid genome assembly of C. fimbriata isolate AS236 was generated. The resulting 31.7-MB assembly was near-chromosome level, with 18 contigs, 6,481 predicted genes, and a BUSCO completion score of 98.4% when compared with the fungus-specific lineage database. Additional Illumina DNA reads from C. manginecans, C. platani, and a second C. fimbriata isolate (C1421) were then mapped to the assembled genome using BOWTIE2 and counted using HTSeq, which identified 148 genes present only within C. fimbriata as molecular diagnostic candidates; 6 single-copy and 35 highly multi-copy (>40 BLAST hits), as determined through a self-BLAST-P alignment. Primers for PCR were designed in the 200-bp flanking region of the first exon for each candidate, and the candidates were validated against a diverse DNA panel containing Ceratocystis species, sweetpotato pathogens, and plants. After validation, two diagnostic candidates amplified only C. fimbriata DNA and were considered to be highly specific to the species. These genetic markers will serve as valuable diagnostic tools with multiple applications including the detection of C. fimbriata in seed, soil, and wash water in sweetpotato production.

Development, Validation, and Utility of Species-Specific Diagnostic Markers for Detection of Peronospora belbahrii.

Peronospora belbahrii is an oomycete and the cause of basil downy mildew, one of the most destructive diseases affecting basil production worldwide. Disease management is challenging due to wind-dispersed sporangia and contaminated seed; therefore, identifying P. belbahrii in seed lots before sale or planting or in the field before symptoms develop could allow for timely deployment of disease management strategies. In this study, a draft genome assembly and next-generation sequencing reads for P. belbahrii, as well as publicly available DNA-seq and RNA-seq reads of several other downy mildew pathogens, were incorporated into a bioinformatics pipeline to predict P. belbahrii-specific diagnostic markers. The specificity of each candidate marker was validated against a diverse DNA collection of P. belbahrii, host tissue, and related oomycetes using PCR. Two species-specific markers were identified and used as templates to develop a highly sensitive probe-based real-time quantitative PCR (qPCR) assay that could detect P. belbahrii in leaf tissue and seed samples. Both markers were capable of reliably detecting as low as 500 fg/µl of P. belbahrii genomic DNA and as few as 10 sporangia. The qPCR assay was then validated with seed samples collected from a basil cultivar experiment. In total, 48 seed samples were collected and tested; P. belbahrii was detected in samples of all cultivars at estimated concentrations of 600 fg/µl up to 250 pg/µl and at as few as 10 sporangia up to >1,000 sporangia. The markers and assays are valuable for diagnostics and identifying P. belbahrii-contaminated seed lots to mitigate the effects of future basil downy mildew epidemics.

Genome Sequencing and Transcriptome Analysis of the Hop Downy Mildew Pathogen Pseudoperonospora humuli Reveal Species-Specific Genes for Molecular Detection.

Pseudoperonospora humuli is an obligate oomycete pathogen of hop (Humulus lupulus) that causes downy mildew, an important disease in most production regions in the Northern Hemisphere. The pathogen can cause a systemic infection in hop, overwinter in the root system, and infect propagation material. Substantial yield loss may occur owing to P. humuli infection of strobiles (seed cones), shoots, and cone-bearing branches. Fungicide application and cultural practices are the primary methods to manage hop downy mildew. However, effective, sustainable, and cost-effective management of downy mildew can be improved by developing early detection systems to inform on disease risk and timely fungicide application. However, no species-specific diagnostic assays or genomic resources are available for P. humuli. The genome of the P. humuli OR502AA isolate was partially sequenced using Illumina technology and assembled with ABySS. The assembly had a minimum scaffold length of 500 bp and an N50 (median scaffold length of the assembled genome) of 19.2 kbp. A total number of 18,656 genes were identified using MAKER standard gene predictions. Additionally, transcriptome assemblies were generated using RNA-seq and Trinity for seven additional P. humuli isolates. Bioinformatics analyses of next generation sequencing reads of P. humuli and P. cubensis (a closely related sister species) identified 242 candidate species-specific P. humuli genes that could be used as diagnostic molecular markers. These candidate genes were validated using polymerase chain reaction against a diverse collection of isolates from P. humuli, P. cubensis, and other oomycetes. Overall, four diagnostic markers were found to be uniquely present in P. humuli. These candidate markers identified through comparative genomics can be used for pathogen diagnostics in propagation material, such as rhizomes and vegetative cuttings, or adapted for biosurveillance of airborne sporangia, an important source of inoculum in hop downy mildew epidemics.

Serum NGAL can act as an early renal safety biomarker during long-term nucleos(t)ide analogue antiviral therapy in chronic hepatitis B.

Tubular renal toxicity is a side-effect of long-term therapy with nucleos(t)ide analogue(s) (NA) in chronic hepatitis B (CHB). There are no established surrogate markers in plasma of early NA-related toxicity. Neutrophil gelatinase-associated lipocalin (NGAL) is a protein produced by tubular cells following renal damage. We aimed therefore to retrospectively compare conventional renal markers (estimated glomerular filtration rates (eGFR) and urinary protein/creatinine ratio uPCR) with a sensitive biomarker (NGAL) in CHB patients on long-term NA therapy and assess the ability of new markers to predict NA-related renal toxicity (new onset of nonalbumin proteinuria). A total of 192 naïve CHB patients (median age 41 years, 78% males, 25% HBeAg+, 35% cirrhosis) were NA treated for at least 5 years (median 8.34 years, range 5.54-11.1 years). The eGFR and uPCR were compared at baseline and last clinical visit with serum NGAL concentrations measured by ELISA at same time-points and assessed according to the presence/absence of nonalbumin proteinuria at last visit. While baseline and last visit eGFR were similar (median:78 vs 84 mL/min), serum NGAL concentrations increased during therapy (median:9.4 vs 16.4 ng/mL, P < .05). The proportion of patients with proteinuria (uPCR > 15) increased between baseline and last visit (4.6% vs 21.4%, P < .05), with 30 (16%) patients having de novo nonalbumin proteinuria at last visit. High baseline NGAL concentrations were exclusive to patients with de novo nonalbumin proteinuria (median:31.7 vs 7.8 ng/mL, P < .01) and baseline NGAL levels >25 mg/mL were predictive of nonalbumin proteinuria at last visit (AUROC = 0.813). In conclusion, serum NGAL can act as a surrogate marker of early renal injury (de novo nonalbumin proteinuria) in CHB on long-term NA therapy.

Mechanism of mda-5 Inhibition by paramyxovirus V proteins.

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.

Effects of testosterone on the transgender male voice.

There is sparse prospective data on the effects of testosterone therapy on the voices of transgender men (also referred to as trans men or female-to-male transsexuals). Our aim was to investigate the timing and degree of voice deepening over 12 months among an ethnically diverse sample of transgender men. This was a prospective 12-month study at an academic outpatient endocrinology clinic and speech and voice center. The participants were seven transgender men naïve to testosterone therapy. All patients received two voice assessments at baseline and one assessment at 3, 6, 9, and 12 months while on intramuscular testosterone esters. Serum testosterone and estradiol concentrations were measured at baseline and every 3 months. All seven transgender men reached a cisgender male mean fundamental frequency (MF0) within 6 months of testosterone therapy and four continued to experience a decrease after 6 months. The mean decrease in MF0 after 12 months of testosterone therapy was 6.4 semitones (49 Hz). Several patterns emerged regarding the extent and timing. For example, some participants showed no decrease in MF0 within the first 3 months of testosterone therapy, whereas others showed their greatest decrease in MF0. We concluded that transgender men who start testosterone therapy display different patterns of voice lowering. Clinicians should counsel transgender men that they may or may not experience voice lowering within the first 3 months of testosterone therapy and that the majority of voice deepening will occur within 6-9 months.

Molecular characterization of Escherichia coli O157:H7 hide contamination routes: feedlot to harvest.

This study was conducted to identify the origin of Escherichia coli O157:H7 contamination on steer hides at the time of harvest. Samples were collected from the feedlot, transport trailers, and packing plant holding pens and from the colons and hides of feedlot steers. A total of 50 hide samples were positive for E. coli O157:H7 in two geographical locations: the Midwest (25 positive hides) and Southwest (25 positive hides). Hide samples were screened, and the presence of E. coli O157: H7 was confirmed. E. coli O157:H7 isolates were fingerprinted by pulsed-field gel electrophoresis and subjected to multiplex PCR procedures for amplification of E. coli O157:H7 genes stx1, stx2, eaeA, fliC, rfbEO157, and hlyA. Feedlot water trough, pen floor, feed bunk, loading chute, truck trailer side wall and floor, packing plant holding pen floor and side rail, and packing plant cattle drinking water samples were positive for E. coli O157:H7. Pulsed-field gel electrophoresis banding patterns were analyzed after classifying isolates according to the marker genes present and according to packing plant. In this study, hide samples positive for E. coli O157:H7 were traced to other E. coli O157:H7-positive hide, colon, feedlot pen floor fecal, packing plant holding pen drinking water, and transport trailer side wall samples. Links were found between packing plant side rails, feedlot loading chutes, and feedlot pens and between truck trailer, different feedlots, and colons of multiple cattle. This study is the first in which genotypic matches have been made between E. coli O157:H7 isolates obtained from transport trailer side walls and those from cattle hide samples within the packing plant.

Differences in QRS configuration during unipolar pacing from adjacent sites: implications for the spatial resolution of pace-mapping.

To examine the spatial resolution of unipolar pace-mapping, 12 lead electrocardiograms (ECGs) recorded during pacing from each of the poles of a quadripolar catheter (5 mm interelectrode distance) were examined. Unipolar pacing was performed from each of the poles at late diastolic threshold, twice threshold and 10 mA at a cycle length of 500 ms. In 15 patients, pacing was performed at the right ventricular apex and in 14 at various left ventricular sites. Pacing from the distal catheter pole at threshold (index ECG) was used to simulate the site of origin of ventricular tachycardia, and all other ECGs were compared with the index ECG. Electrocardiograms were evaluated by two independent observers for 1) minor configuration differences (notch, new small component, change in the amplitude of individual components or change in QRS shape); 2) major differences in configuration (new large component, marked change in the amplitude of an existing component or two minor changes); and 3) peak to peak changes in amplitude. Minor differences in configuration were seen in a mean 2.4 +/- 1.9, 4.6 +/- 2.4 and 4.4 +/- 2.9 leads during pacing at 5, 10 and 15 mm from the distal electrode (index site). Major differences in configuration were seen in a mean of 0.3 +/- 0.5, 2.1 +/- 2.1 and 3.7 +/- 2.3 leads during pacing at 5, 10 and 15 mm from the index site. Differences in amplitude were seen in a mean of 3.1 +/- 2.2, 5.6 +/- 2.5 and 6.8 +/- 3.0 leads per ECG during pacing at 5, 10 and 15 mm from the index ECG pacing site, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes.

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.

Genetic Regulation of Development in Sorghum bicolor (X. Greatly Attenuated Photoperiod Sensitivity in a Phytochrome-Deficient Sorghum Possessing a Biological Clock but Lacking a Red Light-High Irradiance Response).

The role of a light-stable, 123-kD phytochrome in the biological clock, in photoperiodic flowering and shoot growth in extended photoperiods, and in the red light-high irradiance response was studied in Sorghum bicolor using a phytochrome-deficient mutant, 58M (ma3R ma3R), and a near-isogenic wild-type cultivar, 100M (Ma3 Ma3). Since chlorophyll a/b-binding protein mRNA and ribulose bisphosphate carboxylase small subunit mRNA cycled in a circadian fashion in both 58M and 100M grown in constant light, the 123-kD phytochrome absent from 58M does not appear necessary for expression or entrainment of a functional biological clock. Although 58M previously appeared photoperiod insensitive in 12-h photoperiods, extending the photoperiod up to 24 h delayed floral initiation for up to 2 weeks but did not much affect shoot elongation. Thus, although 58M flowers early in intermediate photoperiods, a residual photoperiod sensitivity remains that presumably is not due to the missing 123-kD phytochrome. Since rapid shoot elongation persists in 58M under extended photoperiods despite delayed floral initiation, long photoperiods uncouple those processes. The observed absence of a red light-high irradiance response in 58M, in contrast to the presence of the response in 100M, strengthens the suggestion that the 123-kD phytochrome missing from 58M is a phyB.

Genetic Regulation of Development in Sorghum bicolor (VIII. Shoot Growth, Tillering, Flowering, Gibberellin Biosynthesis, and Phytochrome Levels Are Differentially Affected by Dosage of the ma3R Allele.

Sorghum [Sorghum bicolor (L.) Moench] homozygous for ma3R lacks a type II, light-stable phytochrome of 123 kD and has a number of phenotypic characteristics consistent with the absence of functional phytochrome B. We have used plants heterozygous at Ma3 (Ma3/ma3R and ma3/ma3R) to determine the effect of dosage of ma3R on plant growth, flowering, gibberellin (GA) levels, and content of the 123-kD phytochrome. Both Ma3/ma3R and ma3/ma3R produced the same number of tillers per plant as their respective homozygous non-ma3R parents. Height of the heterozygotes was intermediate between the homozygous parents, although it was more similar to the non-ma3R genotypes. In both field and growth-chamber environments, the timing of floral initiation and anthesis in the heterozygotes also was intermediate, again more similar to non-ma3R plants. In Ma3/ma3R, levels of GA53, GA19, GA20, and GA1 were almost exactly intermediate between levels detected in Ma3/Ma3 and ma3R/ma3R plants. Immunoblot analysis indicated that there was less of the 123-kD phytochrome in Ma3/ma3R than in homozygous Ma3, whereas none was detected in ma3R/ma3R. The degree of dominance of Ma3 and ma3 over ma3R varies with phenotypic trait, indicating that mechanisms of activity of the 123-kD phytochrome vary among the biochemical processes involved in each phenotypic character. Although the heterozygotes were similar to homozygous Ma3 and ma3 plants in growth and flowering behavior, Ma3/ma3R contained 50% less of the bioactive GA (GA1) than non-ma3R genotypes. Thus, sensitivity to endogenous GAs also may be regulated by the 123-kD phytochrome. To fully regulate plant growth and development, two copies of Ma3 or ma3 are required to produce sufficient quantities of the light-stable, 123-kD phytochrome.

Detection of the NS3 Q80K polymorphism by Sanger and deep sequencing in hepatitis C virus genotype 1a strains in the UK.

The Q80K polymorphism in the hepatitis C virus (HCV) NS3 enzyme reduces susceptibility to simeprevir and other novel protease inhibitors. The aims of this study were to determine the prevalence of Q80K in treatment-naïve HCV-1a carriers in the North West region (NW) and South East region (SE) of England, investigate the occurrence of Q80K as a minority variant, and characterize viral phylogeny. Plasma samples from subjects who were naïve to anti-HCV therapy were subjected to conventional (Sanger) and deep (Illumina-Miseq, 1% interpretative cut-off) sequencing of NS3. Q80K occurred in 44 of 238 subjects (18.5%, 95% CI 13.6-23.4%), including 19 of 70 (27.1%) in the NW and 25 of 168 (14.9%) in the SE (p 0.0425), with no difference in HCV RNA load or human immunodeficiency virus (HIV) status. Q80K frequencies in reads of samples subjected to Illumina sequencing were >40% in all cases. Among subjects with Q80K, five of 44 (11.4%) showed one additional major resistance-associated mutation in NS3, detected at frequencies of >10% (V36L and V55A) or <10% (V36M). Phylogenetic analyses identified the two recognized HCV-1a lineages with (clade I) and without (clade II) Q80K. Overall, 148 of 238 (62.2%) sequences occurred within regional or inter-regional clusters, each comprising 3-20 sequences. There was no unique clustering of English sequences relative to strains from continental Europe and North America. In conclusion, Q80K was found at a high prevalence among treatment-naïve HCV-1a carriers in England, and was reliably detected by conventional sequencing, with no increased detection by deep sequencing. English sequences were highly interspersed with sequences from elsewhere in Europe (clade II) and North America (clade I), and their phylogeny was consistent with multiple introductions from different areas.

Intersex in the clam Scrobicularia plana (Da Costa): Widespread occurrence in English Channel estuaries and surrounding areas.

Estuarine clams Scrobicularia plana were sampled from 108 intertidal locations around the English Channel and adjacent areas. Although S. plana is believed to be a strict gonochorist, 58% of the populations sampled included intersexed individuals (described as male clams exhibiting ovotestis). Over the entire region, on average, 8.6% of male clams exhibited intersex, although proportions of affected males ranged from 0% to 53% depending on location. The severity of intersex was assessed using a simple classification scale, with the majority of individuals showing low levels of impact. Sex ratios were significantly skewed at some sites. There were no significant relationships between incidence or severity of intersex; or with size or parasitism of individual clams. Intersex in S. plana is a useful tool to assess endocrine disruptive effects in estuaries, although mechanisms of impact and causative agents remain uncertain.

The use of hibernation induction triggers for cardiac transplant preservation.

Cardiac transplant is hindered by donor shortage and preservation time. Extended extracorporeal preservation could increase the number and distribution of hearts for transplantation. Interestingly, mammalian hibernation biology closely parallels the altered cardiac cellular physiology noted with hypothermic organ storage. The present study undertook to test whether treatment with hibernation induction triggers could improve myocardial functional recovery following prolonged ischemic storage in a nonhibernating mammalian model. To study this hypothesis, isolated rabbit hearts had baseline functional and metabolic parameters recorded and then received either hypothermic storage only or standard cardioplegia, or cardioplegia containing 1 mg/kg D-Ala2-Leu5-enkaphalin (DADLE), which mimics natural hibernation, or preperfusion with DADLE, administered for 15 min at 2 mmol, 25 min prior to cardioplegic ischemia. Hearts were then subjected to 18 hr of global ischemic storage at 4 degrees C. Isovolumic developed pressure, coronary flows, and myocardial oxygen consumption were significantly improved with DADLE pretreatment vs. all groups after storage and reflow. Furthermore, DADLE hearts demonstrated better histological ultrastructure preservation following prolonged storage ischemia. This study demonstrates that hibernation protection with DADLE is beneficial for prolonged cardiac storage. The use of hibernation induction triggers is promising for organ preservation and deserve further mechanistic study.

Flavone improves functional recovery after ischemia in isolated reperfused rabbit hearts.

The effects of flavone (2-phenyl-1,4-benzopyrone), a modulator of the cytochrome P-450 monooxygenase system, on myocardial postischemic reperfusion recovery were examined in the present study. Left ventricular functional recovery was evaluated in isolated, crystalloid-perfused rabbit hearts after 2 hours of modestly hypothermic (34 degrees C) global ischemia. Four groups (n = 8 in each group) were studied and compared: a vehicle control group, a second group pretreated with flavone (8 x 10(-6) mol/L) before ischemia, a third group pretreated with flavone followed by SKF 525-A (1.7 x 10(-5) mol/L), an inhibitor of cytochrome P-450, and a fourth group pretreated with flavone followed by indomethacin (1 x 10(-6) mol/L), an inhibitor of cyclooxygenase. At 15, 30, and 45 minutes after reperfusion, recovery of left ventricular developed pressure in the control group averaged (mean +/- standard deviation) only 2.60% +/- 12.7%, 35.5% +/- 15.0%, and 42.9% +/- 13.5% of baseline, respectively. In the flavone-treated group, recovery was significantly better, averaging 67.7% +/- 10.7%, 73.9% +/- 9.3%, and 73.6% +/- 7.6% of baseline at the same time periods. Recovery of peak positive rate of pressure rise in the control group averaged 27.4% +/- 15.2%, 38.6% +/- 19.2%, and 45.4% +/- 18.6% of baseline at 15, 30, and 45 minutes of reperfusion, respectively. In the flavone-treated group recovery values were significantly higher, averaging 67.8% +/- 9.6%, 77.3% +/- 8.5%, and 77.0% +/- 9.0% of baseline. End-diastolic pressures were significantly lower in the flavone-treated group compared with the control group at all reperfusion time points. Myocardial oxygen consumption was significantly higher in the flavone-treated group at 30 and 45 minutes of reperfusion, as well. The improvement resulting from flavone infusion was abolished completely by SKF 525-A, providing support for the interpretation that the effects of flavone were mediated through the cytochrome P-450 system. The cyclooxygenase inhibitor indomethacin midly attenuated the effects of flavone pretreatment, suggesting that the effects of flavone were only minimally related to metabolites of cyclooxygenase. We conclude that pretreatment with flavone represents a promising approach to myocardial protection that may be due to modulation of the myocardial cytochrome P-450 system.

Enhanced myocardial protection during global ischemia with 5'-nucleotidase inhibitors.

Depletion of adenosine triphosphate precursors, such as myocardial adenosine, during global ischemia results in poor postischemic adenosine triphosphate repletion and functional recovery. Neonatal hearts may be more resistant to this deleterious effect of ischemia, because they are characterized by low 5'-nucleotidase activity, which may result in higher sustained endogenous myocardial adenosine triphosphate precursor levels during ischemia. Adult hearts, however, have high levels of 5'-nucleotidase activity leading to depleted precursors during ischemia and poor postischemic functional recovery. Augmenting myocardial adenosine exogenously during ischemia in adult hearts has a beneficial effect on recovery. The present study tested if preservation of nucleotide precursors, better adenosine triphosphate repletion, and enhanced postischemic myocardial recovery in adult hearts could be achieved with a "neonatal" strategy. Therefore 5'-nucleotidase inhibitors were administered to isolated, perfused adult rabbit hearts subjected to 120 minutes of ischemia (at 34 degrees C) to determine if this improved functional recovery. Hearts received St. Thomas' Hospital cardioplegic solution (control hearts) or cardioplegic solution containing 5'-nucleotidase inhibitors: pentoxifylline, thioinosine, [s-(p-nitrophenyl)-4-thioinosine], or thioinosine's dimethyl sulfoxide vehicle alone. After ischemia and reperfusion, recovery of systolic function, diastolic function, and myocardial oxygen consumption was significantly better with 5'-nucleotidase inhibition. No changes in coronary flow were noted. We speculate and are pursuing the theory that the mechanism of 5'-nucleotidase inhibition's favorable action is due to preventing the catabolism, transport, and loss of nucleotide precursors during ischemia, maintaining adenosine triphosphate precursor availability.

Developmental differences in myocardial protection in response to 5'-nucleotidase inhibition.

Age-related differences in the activity of 5'-nucleotidase, an enzyme responsible for conversion of high-energy phosphates to their the diffusible precursors, may help to explain age-related differences in tolerance of global myocardial ischemia. Postischemic function and high-energy phosphate content were measured in the hearts of rabbits 7 to 10 days old (neonate), 30 to 40 days old (1 month), and 6 to 12 months old (adult). Hearts in each age group were subjected to 60 minutes of ischemia at 34 degrees C either with no cardioplegia, with unmodified St. Thomas' Hospital cardioplegic solution, or with St. Thomas' Hospital cardioplegic solution with pentoxifylline, a 5'-nucleotidase inhibitor. These groups were compared with one another and with control hearts that were continuously perfused for 1 hour. In adults, addition of pentoxifylline to St. Thomas' Hospital cardioplegic solution restored adenosine triphosphate and total nondiffusible nucleotide levels to control values and improved recovery of cardiac output and developed pressure compared with results with unmodified St. Thomas' Hospital cardioplegic solution. In contrast, biochemical and functional parameters in neonatal hearts were not affected by either unmodified St. Thomas' Hospital cardioplegic solution cardioplegia or St. Thomas' Hospital cardioplegic solution with pentoxifylline. Functional recovery in neonatal hearts subjected to unprotected ischemia was superior to that in the older age groups. In 1-month-old hearts, St. Thomas' Hospital cardioplegia improved recovery compared with recovery after unprotected ischemia, but no incremental improvement in function or high-energy stores was seen with addition of pentoxifylline. The lack of effect of pentoxifylline on neonatal hearts suggest that there is a relative deficiency of 5'-nucleotidase in this age group. This may contribute to the improved functional recovery observed in unprotected hearts. Furthermore, addition of pentoxifylline to adult hearts appears to confer the benefits of low 5'-nucleotidase activity occurring naturally in the neonate.

Development and mapping of AFLP markers linked to the sorghum fertility restorer gene rf4.

The restoration of male fertility in the sorghum IS1112 C (A3) male-sterile cytoplasm is through a two-gene gametophytic system involving complementary action of the restoring alleles Rf3 and Rf4. To develop markers suitable for mapping rf4, AFLP technology was applied to bulks of sterile and fertile individuals from a segregating BC(3)F(1) population. Three AFLP markers linked to rf4were identified and subsequently converted to STS/CAPS markers, two of which are co-dominant. Based on a population of 378 BC(1)F(1) individuals, two STS/CAPS markers, LW7 and LW8, mapped to within 5.31 and 3.18 cM, respectively, of rf4, while an STS marker, LW9, was positioned 0.79 cM on the flanking side of rf4. Markers LW8 and LW9 were used to screen sorghum BAC libraries to identify the genomic region encoding rf4. A series of BAC clones shown to represent a genomic region of linkage group E were identified by the rf4-linked markers. A contig of BAC clones flanking the LW9 marker represent seed clones on linkage group E, from which fine mapping of the rf4 locus and chromosome walking can be initiated.

Perfluorocarbon supplementation and postischemic cardiac function.

BACKGROUND: During induced ischemia for cardiac surgery inefficient anaerobic energy mechanisms predominate. Sustaining aerobic metabolism with perfluorocarbon-supplemented blood cardioplegia theoretically could lead to improved postischemic recovery. Therefore we studied functional recovery after myocardial ischemia, comparing perflubron (C8F17Br) supplemented blood cardioplegia to standard blood cardioplegia. METHODS: Nineteen dogs underwent 15 minutes of 37 degrees C global ischemia on cardiopulmonary bypass, followed by 90 minutes of cardioplegic arrest by use of blood cardioplegia with or without perflubron and then 30 minutes of 37 degrees C reperfusion. During ischemia myocardial oxygen tension, temperature, and pH were measured. Postischemic left ventricular recovery was assessed by means of preload recruitable stroke work, exponential end-diastolic stress-strain regression, and preservation of adenosine triphosphate and energy charge. RESULTS: The addition of perflubron, a new shorter half-life, lecithin-emulsified perfluorocarbon, provided superior myocardial protection when compared with standard blood cardioplegia. This benefit was evidenced by significantly increased recovery of preload recruitable stroke work slope (71% +/- 8% versus 42% +/- 9%), decreased myocardial edema, and enhanced end ischemic myocardial oxygen and pH levels. CONCLUSIONS: The reliable oxygen delivery system and endothelial-preserving properties of the perfluorocarbons may prove to be an invaluable asset in addition to standard blood cardioplegia in the preservation of postischemic ventricular function. These data support the further investigation of perfluorocarbon-enriched blood cardioplegia.

Glucose level and myocardial recovery after warm arrest.

BACKGROUND: During induced cold ischemia for cardiac operations, increasing glucose concentration is not thought to enhance myocardial protection and may detrimentally affect recovery. However, during "warm aerobic" arrest, increased glucose availability as substrate could enhance postischemic metabolic and functional recovery, as during and after ischemia, myocytes shift preference for substrate from fatty acids to glucose. Unfortunately, hyperglycemia may also increase patient susceptibility to neurologic injury. METHODS: This experiment was designed to study the optimal dose of glucose and its effect on function during warm arrest. Isolated, retrograde-perfused rabbit hearts received multidose cardioplegia containing increasing concentrations of glucose, from 0 to 88 mmol/L, and underwent 120 minutes of "warm" 34 degrees C global ischemia. Osmolarities were adjusted equivalently. RESULTS: After 34 degrees C ischemia, hearts treated with 5 to 88 mmol/L glucose showed significantly better functional recovery than those treated with 0 to 1 mmol/L glucose. However, the addition of 22 mmol/L glucose demonstrated optimal recovery with no further incremental enhancement with more glucose. Additional hearts receiving 0 or 22 mmol/L glucose had high-energy phosphates, lactate, CO2, and pH measured. The 22 mmol/L glucose hearts demonstrated active metabolism and significantly better recovery of high-energy phosphate levels than controls. CONCLUSIONS: Increasing glucose level modestly during warm arrest enhanced recovery, but profound hyperglycemia did not incrementally improve this effect, mandating a cautious use of glucose.