Browsing by Subject "Polymerase Chain Reaction"

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    A novel mutation of the ACADM gene (c.145C>G) associated with the common c.985A>G mutation on the other ACADM allele causes mild MCAD deficiency: a case report.
    (Orphanet J Rare Dis, 2010-10-05) Dessein, Anne-Frédérique; Fontaine, Monique; Andresen, Brage S; Gregersen, Niels; Brivet, Michèle; Rabier, Daniel; Napuri-Gouel, Silvia; Dobbelaere, Dries; Mention-Mulliez, Karine; Martin-Ponthieu, Annie; Briand, Gilbert; Millington, David S; Vianey-Saban, Christine; Wanders, Ronald JA; Vamecq, Joseph
    A female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial β-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.
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    A recessive variant of XRCC4 predisposes to non- BRCA1/2 breast cancer in chinese women and impairs the DNA damage response via dysregulated nuclear localization.
    (Oncotarget, 2014-12) He, Min; Hu, Xin; Chen, Li; Cao, A-Yong; Yu, Ke-Da; Shi, Ting-Yan; Kuang, Xia-Ying; Shi, Wen-Biao; Ling, Hong; Li, Shan; Qiao, Feng; Yao, Ling; Wei, Qingyi; Di, Gen-Hong; Shao, Zhi-Ming
    XRCC4 plays a crucial role in the non-homologous end joining pathway that maintains genome stability. In this two-stage case-control study with 1,764 non-BRCA1/2 breast cancer patients and 1,623 cancer-free controls, we investigated the contribution of genetic variants of XRCC4 to breast cancer susceptibility in Chinese women. We identified a recessive missense variant, rs3734091 (c.739G>T, p.Ala247Ser), of XRCC4 that was significantly associated with an increased risk of breast cancer (odds ratio [OR] = 3.92, P = 0.007), particularly with the risk of developing triple-negative breast cancer (OR = 18.65, P < 0.0001). This p.Ala247Ser variant disturbed the nuclear localization of XRCC4 in cells homozygous for the rs3734091-T allele but not in heterozygous cells at both the cellular and tissue levels. In heterozygous cells, wild-type XRCC4 facilitated the nuclear localization of the XRCC4A247S mutant, thus compensating for the impaired localization of XRCC4A247S. This provided a biological mechanism by which rs3734091 conferred an increased susceptibility to non-BRCA1/2 breast cancer exclusively under a recessive model. Further functional analyses revealed that p.Ala247Ser impaired the DNA damage repair capacity and ultimately perturbed genomic stability. Taken together, our findings document the role of XRCC4 in non-BRCA1/2 breast cancer predisposition and reveal its underlying biological mechanism of action.
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    CCDC62/ERAP75 functions as a coactivator to enhance estrogen receptor beta-mediated transactivation and target gene expression in prostate cancer cells.
    (Carcinogenesis, 2009-05) Chen, Ming; Ni, Jing; Chang, Hong-Chiang; Lin, Chen-Yong; Muyan, Mesut; Yeh, Shuyuan
    Human prostate cancer (PCa) and prostate epithelial cells predominantly express estrogen receptor (ER) beta, but not ERalpha. ERbeta might utilize various ER coregulators to mediate the E2-signaling pathway in PCa. Here, we identified coiled-coil domain containing 62 (CCDC62)/ERAP75 as a novel ER coactivator. CCDC62/ERAP75 is widely expressed in PCa cell lines and has low expression in MCF7 cells. Both in vitro and in vivo interaction assays using mammalian two-hybrid, glutathione S-transferase pull-down and coimmunoprecipitation methods proved that ERbeta can interact with the C-terminus of CCDC62/ERAP75 via the ligand-binding domain. The first LXXLL motif within CCDC62/ERAP75 is required for the interaction between ERbeta and CCDC62/ERAP75. Electrophoretic mobility shift assay showed that CCDC62/ERAP75 can be recruited by the estrogen response element-ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of CCDC62/ERAP75 within the promoter of the estrogen-responsive gene cyclin D1. In addition, using silencing RNA (siRNA) against endogeneous CCDC62/ERAP75, we demonstrated that inhibition of endogenous CCDC62/ERAP75 results in the suppression of ERbeta-mediated transactivation as well as target gene expression in LNCaP cells. More importantly, using the tet-on overexpression system, we showed that induced expression of CCDC62/ERAP75 can enhance the E2-regulated cyclin D1 expression and cell growth in LNCaP cells. Together, our results revealed the role of CCDC62/ERAP75 as a novel coactivator in PCa cells that can modulate ERbeta transactivation and receptor function.
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    Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal.
    (PLoS Pathog, 2014-08) Springer, DJ; Billmyre, RB; Filler, EE; Voelz, K; Pursall, R; Mieczkowski, PA; Larsen, RA; Dietrich, FS; May, RC; Filler, SG; Heitman, J
    Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.
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    Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns.
    (BMC evolutionary biology, 2007-09-08) Haugen, P; Bhattacharya, D; Palmer, JD; Turner, S; Lewis, LA; Pryer, KM
    Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria.Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG), in large subunit (LSU) rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria.We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene). The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.
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    Delimiting species without nuclear monophyly in Madagascar's mouse lemurs.
    (PLoS One, 2010-03-31) Weisrock, David W; Rasoloarison, Rodin M; Fiorentino, Isabella; Ralison, José M; Goodman, Steven M; Kappeler, Peter M; Yoder, Anne D
    BACKGROUND: Speciation begins when populations become genetically separated through a substantial reduction in gene flow, and it is at this point that a genetically cohesive set of populations attain the sole property of species: the independent evolution of a population-level lineage. The comprehensive delimitation of species within biodiversity hotspots, regardless of their level of divergence, is important for understanding the factors that drive the diversification of biota and for identifying them as targets for conservation. However, delimiting recently diverged species is challenging due to insufficient time for the differential evolution of characters--including morphological differences, reproductive isolation, and gene tree monophyly--that are typically used as evidence for separately evolving lineages. METHODOLOGY: In this study, we assembled multiple lines of evidence from the analysis of mtDNA and nDNA sequence data for the delimitation of a high diversity of cryptically diverged population-level mouse lemur lineages across the island of Madagascar. Our study uses a multi-faceted approach that applies phylogenetic, population genetic, and genealogical analysis for recognizing lineage diversity and presents the most thoroughly sampled species delimitation of mouse lemur ever performed. CONCLUSIONS: The resolution of a large number of geographically defined clades in the mtDNA gene tree provides strong initial evidence for recognizing a high diversity of population-level lineages in mouse lemurs. We find additional support for lineage recognition in the striking concordance between mtDNA clades and patterns of nuclear population structure. Lineages identified using these two sources of evidence also exhibit patterns of population divergence according to genealogical exclusivity estimates. Mouse lemur lineage diversity is reflected in both a geographically fine-scaled pattern of population divergence within established and geographically widespread taxa, as well as newly resolved patterns of micro-endemism revealed through expanded field sampling into previously poorly and well-sampled regions.
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    Detection of Bartonella species in the blood of veterinarians and veterinary technicians: a newly recognized occupational hazard?
    (Vector Borne Zoonotic Dis, 2014-08) Lantos, Paul M; Maggi, Ricardo G; Ferguson, Brandy; Varkey, Jay; Park, Lawrence P; Breitschwerdt, Edward B; Woods, Christopher W
    BACKGROUND: Bartonella species are important emerging pathogens in human and veterinary medicine. In the context of their daily activities, veterinary professionals have frequent animal contact and arthropod exposures. Detection of Bartonella spp. using traditional culture methods has been limited by poor sensitivity, making it difficult to determine the prevalence of infection in this population. We have developed a detection method combining enrichment culture and molecular amplification, which increases testing sensitivity. METHODS: We performed a cross-sectional study to determine the prevalence of detectable Bartonella spp. in the blood of veterinary personnel and nonveterinary control subjects. Bartonella was detected by enrichment blood culture with conventional PCR followed by DNA sequencing. RESULTS were correlated with epidemiological variables and symptoms. RESULTS: We detected DNA from at least one Bartonella species in 32 (28%) of the 114 veterinary subjects. After DNA sequencing, the Bartonella species could be determined for 27 of the 32 infected subjects, including B. henselae in 15 (56%), B. vinsonii subsp. berkhoffii in seven (26%), B. koehlerae in six (22%), and a B. volans-like sequence in one (4%). Seventy percent of Bartonella-positive subjects described headache compared with 40% of uninfected veterinarians (p=0.009). Irritability was also reported more commonly by infected subjects (68% vs. 43%, p=0.04). CONCLUSIONS: Our study supports an emerging body of evidence that cryptic Bartonella bloodstream infection may be more frequent in humans than previously recognized and may induce symptoms. Longitudinal studies are needed to determine the natural course and clinical features of Bartonella infection.
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    Dual infection with Ehrlichia chaffeensis and a spotted fever group rickettsia: a case report.
    (Emerging infectious diseases, 1998-04) Sexton, DJ; Corey, GR; Carpenter, C; Kong, LQ; Gandhi, T; Breitschwerdt, E; Hegarty, B; Chen, SM; Feng, HM; Yu, XJ; Olano, J; Walker, DH; Dumler, SJ
    Well-documented cases of simultaneous human infection with more than one tick-borne pathogen are rare. To our knowledge only two dual infections have been reported: simultaneous human infection with the agent of human granulocytic ehrlichiosis and Borrelia burgdorferi and simultaneous human infection with B. burgdorferi and Babesia microti (1-2). Rocky Mountain spotted fever has long been known to be endemic in North Carolina; cases of human ehrlichial infection were recognized there soon after Ehrlichia chaffeensis was recognized as an important cause of tick-borne disease in the southeastern United States. Because both Rocky Mountain spotted fever and ehrlichiosis are prevalent in North Carolina, occasional cases of simultaneous human infection by rickettsial and ehrlichial agents would not be surprising; however, no such cases seem to have been reported.
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    Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.
    (PLoS Pathog, 2010-04-22) Byrnes 3rd, EJ; Li, W; Lewit, Y; Ma, H; Voelz, K; Ren, P; Carter, DA; Chaturvedi, V; Bildfell, RJ; May, RC; Heitman, J
    Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.
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    Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function.
    (Proc Natl Acad Sci U S A, 1996-11-12) Jaber, M; Koch, WJ; Rockman, H; Smith, B; Bond, RA; Sulik, KK; Ross, J; Lefkowitz, RJ; Caron, MG; Giros, B
    The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a > 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.
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    Evidence that SOX2 overexpression is oncogenic in the lung.
    (PLoS One, 2010-06-10) Lu, Y; Futtner, C; Rock, JR; Xu, X; Whitworth, W; Hogan, BL; Onaitis, MW
    BACKGROUND: SOX2 (Sry-box 2) is required to maintain a variety of stem cells, is overexpressed in some solid tumors, and is expressed in epithelial cells of the lung. METHODOLOGY/PRINCIPAL FINDINGS: We show that SOX2 is overexpressed in human squamous cell lung tumors and some adenocarcinomas. We have generated mouse models in which Sox2 is upregulated in epithelial cells of the lung during development and in the adult. In both cases, overexpression leads to extensive hyperplasia. In the terminal bronchioles, a trachea-like pseudostratified epithelium develops with p63-positive cells underlying columnar cells. Over 12-34 weeks, about half of the mice expressing the highest levels of Sox2 develop carcinoma. These tumors resemble adenocarcinoma but express the squamous marker, Trp63 (p63). CONCLUSIONS: These findings demonstrate that Sox2 overexpression both induces a proximal phenotype in the distal airways/alveoli and leads to cancer.
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    Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.
    (Proc Natl Acad Sci U S A, 1990-07) Lomasney, JW; Lorenz, W; Allen, LF; King, K; Regan, JW; Yang-Feng, TL; Caron, MG; Lefkowitz, RJ
    Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.
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    From offshore to onshore: multiple origins of shallow-water corals from deep-sea ancestors.
    (PLoS One, 2008-06-18) Lindner, Alberto; Cairns, Stephen D; Cunningham, Clifford W
    Shallow-water tropical reefs and the deep sea represent the two most diverse marine environments. Understanding the origin and diversification of this biodiversity is a major quest in ecology and evolution. The most prominent and well-supported explanation, articulated since the first explorations of the deep sea, holds that benthic marine fauna originated in shallow, onshore environments, and diversified into deeper waters. In contrast, evidence that groups of marine organisms originated in the deep sea is limited, and the possibility that deep-water taxa have contributed to the formation of shallow-water communities remains untested with phylogenetic methods. Here we show that stylasterid corals (Cnidaria: Hydrozoa: Stylasteridae)--the second most diverse group of hard corals--originated and diversified extensively in the deep sea, and subsequently invaded shallow waters. Our phylogenetic results show that deep-water stylasterid corals have invaded the shallow-water tropics three times, with one additional invasion of the shallow-water temperate zone. Our results also show that anti-predatory innovations arose in the deep sea, but were not involved in the shallow-water invasions. These findings are the first robust evidence that an important group of tropical shallow-water marine animals evolved from deep-water ancestors.
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    In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells.
    (J Exp Med, 1993-10-01) Jacob, J; Przylepa, J; Miller, C; Kelsoe, G
    In the murine spleen, germinal centers are the anatomic sites for antigen-driven hypermutation and selection of immunoglobulin (Ig) genes. To detail the kinetics of Ig mutation and selection, 178 VDJ sequences from 16 antigen-induced germinal centers were analyzed. Although germinal centers appeared by day 4, mutation was not observed in germinal center B cells until day 8 postimmunization; thereafter, point mutations favoring asymmetrical transversions accumulated until day 14. During this period, strong phenotypic selection on the mutant B lymphocytes was inferred from progressively biased distributions of mutations within the Ig variable region, the loss of crippling mutations, decreased relative clonal diversity, and increasingly restricted use of canonical gene segments. The period of most intense selection on germinal center B cell populations preceded significant levels of mutation and may represent a physiologically determined restriction on B cells permitted to enter the memory pathway. Noncanonical Ig genes recovered from germinal centers were mostly unmutated although they probably came from antigen-reactive cells. Together, these observations demonstrate that the germinal center microenvironment is rich and temporally complex but may not be constitutive for somatic hypermutation.
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    Interactions between life stress factors and carrying the APOE4 allele adversely impact self-reported health in old adults.
    (The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2011-10) Zeng, Yi; Hughes, Claude L; Lewis, Megan A; Li, Jianxin; Zhang, Fengyu
    Based on the multiple logistic regression analysis of data from a random sample of 1,023 old adults collected in Taiwan in 2000, we found that interactions between carrying the APOE4 allele and one of four life stress factors (relocated mainlander, living in a crowded household with six or more persons, living in an earthquake-damaged house, and monthly financial difficulty) significantly increased the odds ratio of poor self-reported health. Correlations between carrying the APOE4 allele and the life stress factors were ruled out by statistical tests. These life stress factors had a substantially larger adverse impact on self-reported health in APOE4 allele carriers than in noncarriers. This study provides evidence that interaction between carrying APOE4 allele and chronic life stressors has significant impacts on self-reported health while controlling for various sociodemographic and health behavior factors. Further studies with richer biomarkers are warranted for deeper understanding of the biological mechanisms.
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    Invasive aspergillosis in critically ill patients: Review of definitions and diagnostic approaches.
    (Mycoses, 2021-09) Jenks, Jeffrey D; Nam, Hannah H; Hoenigl, Martin
    Invasive aspergillosis (IA) is an increasingly recognised phenomenon in critically ill patients in the intensive care unit, including in patients with severe influenza and severe coronavirus disease 2019 (COVID-19) infection. To date, there are no consensus criteria on how to define IA in the ICU population, although several criteria are used, including the AspICU criteria and new consensus criteria to categorise COVID-19-associated pulmonary aspergillosis (CAPA). In this review, we describe the epidemiology of IA in critically ill patients, most common definitions used to define IA in this population, and most common clinical specimens obtained for establishing a mycological diagnosis of IA in the critically ill. We also review the most common diagnostic tests used to diagnose IA in this population, and lastly discuss the most common clinical presentation and imaging findings of IA in the critically ill and discuss areas of further needed investigation.
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    Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens.
    (Diagn Microbiol Infect Dis, 2010-05) Wulff-Burchfield, Elizabeth; Schell, Wiley A; Eckhardt, Allen E; Pollack, Michael G; Hua, Zhishan; Rouse, Jeremy L; Pamula, Vamsee K; Srinivasan, Vijay; Benton, Jonathan L; Alexander, Barbara D; Wilfret, David A; Kraft, Monica; Cairns, Charles B; Perfect, John R; Mitchell, Thomas G
    Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.
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    Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.
    (J Neurosurg Spine, 2014-09) Karikari, Isaac O; Gilchrist, Christopher L; Jing, Liufang; Alcorta, David A; Chen, Jun; Richardson, William J; Gabr, Mostafa A; Bell, Richard D; Kelley, Michael J; Bagley, Carlos A; Setton, Lori A
    OBJECT: Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. METHODS: Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups. RESULTS: The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2-3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1-2) with a predominantly heterogeneous staining pattern. CONCLUSIONS: This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.
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    Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.
    (PLoS One, 2010-03-10) Rodriguez-Carres, Marianela; Findley, Keisha; Sun, Sheng; Dietrich, Fred S; Heitman, Joseph
    The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.