766 research outputs found

    Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

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    DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with 60Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone

    Using EuGeneCiD and EuGeneCiM computational tools for synthetic biology

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    Synthetic biology often relies on the design of genetic circuits, utilizing ‘‘bio parts’’ (modular DNA pieces) to accomplish desired responses to external stimuli. While such designs are usually intuited, detailed here is a computational approach to synthetic biology design and modeling using optimization-based tools named Eukaryotic Genetic Circuit Design and Modeling. These allow for designing and subsequent screening of genetic circuits to increase the chances of in vivo success and contribute to the development of an application development pipeline. For complete details on the use and execution of this protocol, please refer to Schroeder, Baber, and Saha (2021)

    A urinary Common Rejection Module (uCRM) score for non-invasive kidney transplant monitoring.

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    A Common Rejection Module (CRM) consisting of 11 genes expressed in allograft biopsies was previously reported to serve as a biomarker for acute rejection (AR), correlate with the extent of graft injury, and predict future allograft damage. We investigated the use of this gene panel on the urine cell pellet of kidney transplant patients. Urinary cell sediments collected from patients with biopsy-confirmed acute rejection, borderline AR (bAR), BK virus nephropathy (BKVN), and stable kidney grafts with normal protocol biopsies (STA) were analyzed for expression of these 11 genes using quantitative polymerase chain reaction (qPCR). We assessed these 11 CRM genes for their abundance, autocorrelation, and individual expression levels. Expression of 10/11 genes were elevated in AR when compared to STA. Psmb9 and Cxcl10could classify AR versus STA as accurately as the 11-gene model (sensitivity = 93.6%, specificity = 97.6%). A uCRM score, based on the geometric mean of the expression levels, could distinguish AR from STA with high accuracy (AUC = 0.9886) and correlated specifically with histologic measures of tubulitis and interstitial inflammation rather than tubular atrophy, glomerulosclerosis, intimal proliferation, tubular vacuolization or acute glomerulitis. This urine gene expression-based score may enable the non-invasive and quantitative monitoring of AR

    Examining the role of menthol cigarettes in progression to established T smoking among youth

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    Background: Menthol, a flavoring compound added to cigarettes, makes cigarettes more appealing to youth and inexperienced smokers and increases cigarettes\u27 abuse liability. However, limited studies are available on menthol\u27s role in smoking progression. Methods: To assess the association between menthol in cigarettes and progression to established smoking, we used five waves of data from the Evaluation of Public Education Campaign on Teen Tobacco Cohort Study, a nationally representative longitudinal survey of U.S. youth conducted as part of “The Real Cost” evaluation. We used discrete time survival analysis to model the occurrence of two event outcomes—progression to established, current smoking and progression to established, frequent smoking—using a logit model with a menthol use indicator as the key explanatory variable. Based on this framework, we estimated the effect of prior menthol use on the odds of smoking progression. Results: In the progression to established, current smoking model, prior menthol use was significantly associated with progression [adjusted odds ratio (aOR) = 1.80, p \u3c .05, confidence interval (CI) = (1.03–3.16)]. While results were in a similar direction for the model of progression to established, frequent smoking, the association between prior menthol use and this progression model did not reach significance [aOR=1.56, CI = (0.80–3.03)]. Conclusion: The results suggest a relationship between using menthol cigarettes and progression from experi- mental to established, current smoking among youth. This study adds to a growing literature base that supports that menthol cigarettes, compared to nonmenthol cigarettes, put youth at increased risk for regular cigarette use

    Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K+ Channels

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    Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels. Includes Supporting Informatio

    Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K+ Channels

    Get PDF
    Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels. Includes Supporting Informatio
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