Douglas D. Rhoads

Pulmonary arterial hypertension (ascites syndrome) in broilers: A review

Pulmonary arterial hypertension (PAH) syndrome in broilers (also known as ascites syndrome and pulmonary hypertension syndrome) can be attributed to imbalances between cardiac output and the anatomical capacity of the pulmonary vasculature to accommodate ever-increasing rates of blood flow, as well as to an inappropriately elevated tone (degree of constriction) maintained by the pulmonary arterioles. Comparisons of PAH-susceptible and PAH-resistant broilers do not consistently reveal differences in cardiac output, but PAH-susceptible broilers consistently have higher pulmonary arterial pressures and pulmonary vascular resistances compared with PAH-resistant broilers. Efforts clarify the causes of excessive pulmonary vascular resistance have focused on evaluating the roles of chemical mediators of vasoconstriction and vasodilation, as well as on pathological (structural) changes occurring within the pulmonary arterioles (e.g., vascular remodeling and pathology) during the pathogenesis of PAH. The objectives of this review are to (1) summarize the pathophysiological progression initiated by the onset of pulmonary hypertension and culminating in terminal ascites; (2) review recent information regarding the factors contributing to excessively elevated resistance to blood flow through the lungs; (3) assess the role of the immune system during the pathogenesis of PAH; and (4) present new insights into the genetic basis of PAH. The cumulative evidence attributes the elevated pulmonary vascular resistance in PAH-susceptible broilers to an anatomically inadequate pulmonary vascular capacity, to excessive vascular tone reflecting the dominance of pulmonary vasoconstrictors over vasodilators, and to vascular pathology elicited by excessive hemodynamic stress. Emerging evidence also demonstrates that the pathogenesis of PAH includes characteristics of an inflammatory/autoimmune disease involving multifactorial genetic, environmental, and immune system components. Pulmonary arterial hypertension susceptibility appears to be multigenic and may be manifested in aberrant stress sensitivity, function, and regulation of pulmonary vascular tissue components, as well as aberrant activities of innate and adaptive immune system components. Major genetic influences and high heritabilities for PAH susceptibility have been demonstrated by numerous investigators. Selection pressures rigorously focused to challenge the pulmonary vascular capacity readily expose the genetic basis for spontaneous PAH in broilers. Chromosomal mapping continues to identify regions associated with ascites susceptibility, and candidate genes have been identified. Ongoing immunological and genomic investigations are likely to continue generating important new knowledge regarding the fundamental biological bases for the PAH/ascites syndrome.

Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs.

The Chinese hamster ovary (CHO) cell 40S ribosomal subunit protein S14 provides a unique opportunity to investigate an important mammalian housekeeping gene and its mRNA and protein products. The S14 gene appears to be single copy, and CHO cell S14 mutants have been isolated as emetine-resistant (emtB) clones in tissue culture. Thus, S14 is the only mammalian ribosomal protein whose gene structure and function are amenable to straightforward genetic and biochemical analysis. Recently, we isolated a wild-type Chinese hamster lung cell cDNA clone, pCS14-1, including an almost complete copy of the ribosomal protein S14 message (N. Nakamichi, D. D. Rhoads, and D. J. Roufa, J. Biol. Chem. 258: 13236-13242, 1983). Here we describe comparable cDNAs from wild-type and emtB CHO cells. We report both mRNA and polypeptide sequences of the wild-type and mutant ribosomal protein transcripts. As a consequence of the genetic methods used to obtain our emetine-resistant mutants, the emtB S14 cDNAs differ from wild-type cDNA by single-base changes. Physical and chemical features of polypeptides encoded by the cDNAs are consistent with well-characterized S14 protein polymorphisms. The three emtB mutations analyzed affect two adjacent arginine codons within the very basic S14 carboxyl region, indicating a significant role for this portion of the protein in the function and architecture of the mammalian 40S ribosomal subunit.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: A proteome-based model for sperm mobility phenotype

Sperm mobility is defined as sperm movement against resistance at body temperature. Although all mobile sperm are motile, not all motile sperm are mobile. Sperm mobility is a primary determinant of male fertility in the chicken. Previous work explained phenotypic variation at the level of the sperm cell and the mitochondrion. The present work was conducted to determine if phenotypic variation could be explained at the level of the proteome using semen donors from lines of chickens selected for low or high sperm mobility. We began by testing the hypothesis that premature mitochondrial failure, and hence sperm immobility, arose from Ca(2+) overloading. The hypothesis was rejected because staining with a cell permeant Ca(2+)-specific dye was not enhanced in the case of low mobility sperm. The likelihood that sperm require little energy before ejaculation and the realization that the mitochondrial permeability transition can be induced by oxidative stress arising from inadequate NADH led to the hypothesis that glycolytic enzymes might differ between lines. This possibility was confirmed by 2-dimensional electrophoresis for aldolase and phosphoglycerate kinase 1. This outcome warranted evaluation of the whole cell proteome by differential detergent fractionation and mass spectrometry. Bioinformatics evaluation of proteins with different expression levels confirmed the likelihood that ATP metabolism and glycolysis differ between lines. This experimental outcome corroborated differences observed between lines in previous work, which include mitochondrial ultrastructure, sperm cell oxygen consumption, and straight line velocity. Although glycolytic proteins were more abundant within highly mobile sperm, quantitative PCR of representative testis RNA, which included mRNA for phosphoglycerate kinase 1, found no difference between lines. In summary, we propose a proteome-based model for sperm mobility phenotype in which a genetic predisposition puts sperm cells at risk of premature mitochondrial failure as they pass through the excurrent ducts of the testis. In other words, we attribute mitochondrial failure to sperm cell and reproductive tract attributes that interact to affect sperm in a stochastic manner before ejaculation. In conclusion, our work provides a starting point for understanding chicken semen quality in terms of gene networks.

GENETIC VARIATION IN BLACK BEARS IN ARKANSAS AND LOUISIANA USING MICROSATELLITE DNA MARKERS

Abstract In the 1950s and 1960s, translocation projects reintroduced black bears (Ursus americanus) from Minnesota and Manitoba to Arkansas and Louisiana. Today, several geographically disconnected populations exist in Arkansas and Louisiana, but their origins are unclear. Some populations may represent a separate subspecies, U. a. luteolus, which is federally protected. We characterized 5 microsatellite loci in 5 isolated populations in Arkansas and Louisiana and compared them with genotypes from Minnesota. Our data indicate that bears of the Ozark and Ouachita mountains of Arkansas, an inland area of Louisiana, and those of Minnesota are similar in overall genetic diversity and allele frequencies, consistent with these populations being wholly or mostly descended from bears from the reintroduction programs. In contrast, bears from southeastern Arkansas and the coastal region of Louisiana genetically are more restricted and homogeneous. Because they exhibit a limited set of genotypes found in the other black bear populations, they represent isolated fragments of a single North American black bear population. Furthermore, genetic distance estimates indicate that the bears in southeastern Arkansas are more genetically distinct from bears in Louisiana, which are currently federally protected.

Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster.

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

Plexiform Lesions in the Lungs of Domestic Fowl Selected for Susceptibility to Pulmonary Arterial Hypertension: Incidence and Histology

Plexiform lesions develop in the pulmonary arteries of humans suffering from idiopathic pulmonary arterial hypertension (IPAH). Plexogenic arteriopathy rarely develops in existing animal models of IPAH. In this study, plexiform lesions developed in the lungs of rapidly growing meat‐type chickens (broiler chickens) that had been genetically selected for susceptibility to IPAH. Plexiform lesions developed spontaneously in: 42% of females and 40% of males; 35% of right lungs, and 45% of left lungs; and, at 8, 12, 16, 20, 24, and 52 weeks of age the plexiform lesion incidences averaged 52%, 50%, 51%, 40%, 36%, and 22%, respectively. Plexiform lesions formed distal to branch points in muscular interparabronchial pulmonary arteries exhibiting intimal proliferation. Perivascular mononuclear cell infiltrates consistently surrounded the affected arteries. Proliferating intimal cells fully or partially occluded the arterial lumen adjacent to plexiform lesions. Broilers reared in clean stainless steel cages exhibited a 50% lesion incidence that did not differ from the 64% incidence in flock mates grown on dusty floor litter. Microparticles (30 μm diameter) were injected to determine if physical occlusion and focal inflammation within distal pulmonary arteries might initiate plexiform lesion development. Three months postinjection no plexiform lesions were observed in the vicinity of persisting microparticles. Broiler chickens selected for innate susceptibility to IPAH represent a new animal model for investigating the mechanisms responsible for spontaneous plexogenic arteriopathy. Anat Rec, 2011.

Efficient separation and sensitive detection of Listeria monocytogenes using an impedance immunosensor based on magnetic nanoparticles, a microfluidic chip, and an interdigitated microelectrode.

Listeria monocytogenes continues to be a major foodborne pathogen that causes food poisoning, and sometimes death, among immunosuppressed people and abortion among pregnant women. In this study, magnetic nanoparticles with a diameter of 30 nm were functionalized with anti-L. monocytogenes antibodies via biotin-streptavidin bonds to become immunomagnetic nanoparticles (IMNPs) to capture L. monocytogenes in a sample during a 2-h immunoreaction. A magnetic separator was used to collect and hold the IMNPs-L. monocytogenes complex while the supernatants were removed. After the washing step, the nanoparticle-L. monocytogenes complex was separated from the sample and injected into a microfluidic chip. The impedance change caused by L. monocytogenes was measured by an impedance analyzer through the interdigitated microelectrode in the microfluidic chip. For L. monocytogenes in phosphate-buffered saline solution, up to 75% of the cells in the sample could be separated, and as few as three to five cells in the microfluidic chip could be detected, which is equivalent to 10(3) CFU/ml of cells in the original sample. The detection of L. monocytogenes was not interfered with by other major foodborne bacteria, including E. coli O157:H7, E. coli K-12, L. innocua, Salmonella Typhimurium, and Staphylococcus aureus. A linear correlation (R(2) = 0.86) was found between the impedance change and the number of L. monocytogenes in a range of 10(3) to 10(7) CFU/ml. Equivalent circuit analysis indicated that the impedance change was mainly due to the decrease in medium resistance when the IMNPs-L. monocytogenes complexes existed in mannitol solution. Finally, the immunosensor was evaluated with food sample tests; the results showed that, without preenrichment and labeling, 10(4) and 10(5) CFU/ml L. monocytogenes in lettuce, milk, and ground beef samples could be detected in 3 h.

A cloned human ribosomal protein gene functions in rodent cells

Cloned fragments of human ribosomal protein S14 DNA (RPS14) were transfected into cultured Chinese hamster (CHO) cells. Transient expression assays indicated that DNA with as little as 31 base pairs of upstream flanking sequence was transcribed into a polyadenylated, 650-base mRNA that is largely bound to the polyribosomes. In these respects the exogenous human S14 message appeared to function normally in CHO cells. Interestingly, transcription of human RPS14 did not require the TATA sequence located 26 base pairs upstream of exon 1. Stably transformed clones were selected from cultures of emetine-resistant CHO cells (Emr-2) after transfection with pSV2Neo-human RPS14 constructs. Human RPS14 complemented the mutationally based drug resistance of the Chinese hamster cells, demonstrating that the cloned human ribosomal protein gene is functional in rodent cells. Analysis of transformed cells with different amounts of integrated RPS14 indicated that human S14 mRNA levels are not tightly regulated by CHO cells. In contrast, the steady-state S14 level fluctuated only slightly, if at all, in transformed clones whose S14 message contents differed by more than 30-fold. These data support the conclusion that expression of human RPS14 is regulated, at least partially, posttranscriptionally.

Multiple regulatory elements ensure accurate transcription of a human ribosomal protein gene

Previously we have shown that expression of a cloned human ribosomal protein gene,RPS14, depends upon regulatory sites located within the genes proximal upstream DNA plus its first intron. In order to identifycis-active sequence motifs within theRPS14 promoter-enhancer complex, we transiently expressed a set of informative deletion clones in cultured Chinese hamster ovary cells. These experiments revealed three DNA sequence motifs that surround the S14 mRNA initiation site and are necessary for accurate transcription. Electrophoretic mobility shift, DNase I footprint, and methylation interference assays resolved two nuclear proteins, NFα-1 and NFβ-1, which bind specifically to these regulatory motifs. NF-α1 recognizes a pair of 6-bp target motifs (5′-TTCCGG-3′) that flank the 5′ end ofRPS14 exon I; and NF-β1 binds to a 10-bp target sequence (5′-CCGTGGGAAC-3′) within the genes first intron. Site-directed deletion mutations within the NF-α1 and -β1 binding sites markedly inhibit S14 mRNA transcription.

An investigation into blood microbiota and its potential association with Bacterial Chondronecrosis with Osteomyelitis (BCO) in Broilers.

Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of lameness in commercial broiler chickens worldwide. BCO represents substantial production loss and welfare issues of chickens. The bacterial species or communities underlying BCO pathogenesis still remain to be fully characterized. To gain insights on blood microbiota in broilers and its potential association with BCO, blood samples collected from healthy (n = 240) and lame (n = 12) chickens were analyzed by deep sequencing of 16S RNA genes. The chicken blood microbiota were dominated by Proteobacteria (60.58% ± 0.65) followed by Bactroidetes (13.99% ± 0.29), Firmicutes (11.45% ± 0.51), Actinobacteria (10.21% ± 0.37) and Cyanobacteria (1.96% ± 0.21) that constituted 98.18% (± 0.22) of the whole phyla. The bacterial communities consist of 30–40 OTUs in the blood of broiler chickens, regardless of ages and other environmental or host conditions, and the blood microbiomes of BCO chickens were largely distinct from those of healthy chickens. In addition, Linear discriminant analysis (LDA) effect size (LEfSe) method revealed that Staphylococcus, Granulicatella, and Microbacterium were significantly enriched in BCO chickens as compared to healthy chickens. The results from this study have significant implications in understanding blood microbiota present in broiler chickens and its potential role in BCO pathogenesis.