The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing ...in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.
Introduction: Non-invasive assays are needed to better discriminate patients with prostate cancer (PCa) to avoid over-treatment of indolent disease. We analyzed 14 methylated DNA markers (MDMs) from ...urine samples of patients with biopsy-proven PCa relative to healthy controls and further studied discrimination of clinically significant PCa (csPCa) from healthy controls and Gleason 6 cancers. Methods: To evaluate the panel, urine from 24 healthy male volunteers with no clinical suspicion for PCa and 24 men with biopsy-confirmed disease across all Gleason scores was collected. Blinded to clinical status, DNA from the supernatant was analyzed for methylation signal within specific DNA sequences across 14 genes (HES5, ZNF655, ITPRIPL1, MAX.chr3.6187, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.8028, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. Results: Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel resulted in 71% sensitivity (95% CI: 49–87%) for PCa detection (4/7 Gleason 6, 8/12 Gleason 7, 5/5 Gleason 8+) and 76% (50–92%) for csPCa (Gleason ≥ 7). At 100% specificity for controls and Gleason 6 patients combined, MDM panel sensitivity was 59% (33–81%) for csPCa (5/12 Gleason 7, 5/5 Gleason 8+). Conclusions: MDMs assayed in urine offer high sensitivity and specificity for detection of clinically significant prostate cancer. Prospective evaluation is necessary to estimate discrimination of patients as first-line screening and as an adjunct to prostate-specific antigen (PSA) testing.
Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. ...However, little is known about how non-essential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a non-essential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5' splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing non-consensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the 1st-step catalytic site, promoting 2nd-step catalysis, and permiting alternate 5' SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.
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Background: Controversy surrounding prostate-specific antigen (PSA) based screening for prostate cancer (PCa) highlights the need for non-invasive assays that better discriminate patients with ...and without clinically significant prostate cancer (csPCa). Such distinction avoids overtreatment in patients with absent or indolent disease while capturing pathology that would derive benefit from intervention. We analyzed the presence of specific methylated DNA markers (MDMs) within urine samples of patients with biopsy proven PCa and assessed the ability to discriminate these patients from healthy controls with no clinical suspicion for PCa. Methods: 24 healthy volunteers with no clinical suspicion for PCa were age-matched to 24 patients with biopsy-confirmed disease across all Gleason scores. Urine collected from subjects was centrifuged with the cell-free supernatant analyzed in a blinded fashion for methylation signal within specific DNA sequences across 14 genes ( HES5, ZNF655, ITPRIPL1, MAX.chr3.193, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.727, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. A patient was considered to have a positive MDM panel if any individual marker exceeded the corresponding100% specificity cut-off value (overall MDM panel specificity of 100%). MDM panel positivity was used to evaluate the sensitivity for distinguishing patients with PCa (any Gleason score) from controls as well as discriminate csPCa cancers from PCa Gleason 6. Results: Median age of healthy controls and PCa patients was 70 years (IQR 67-72) and 65 years (IQR 61-71), respectively. Median PSA was 6.4 (IQR 4.9-9.0) among PCa patients, including median PSA of 5 (IQR 4.0-6.1) for Gleason 6, and 7.8 (IQR 5.1-9.2) for Gleason ≥7 disease. Gleason 6, Gleason 7, and Gleason 8+ cancer was noted in 8, 10, and 6 patients, respectively. Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel revealed an overall sensitivity of 83% (95% CI: 63-95%) for detection of PCa (6 of 8 Gleason 6, 9 of 10 Gleason 7, 5 of 6 Gleason 8+) and 88% (95% CI: 62-98%) for csPCa (Gleason ≥ 7). When considering a 100% specificity threshold for controls and Gleason 6 patients, the sensitivity the MDM panel was 69% (95% CI: 41-89%) for csPCa (6 of 10 Gleason 7, 5 of 6 Gleason 8+). Conclusions: We describe a panel of 14 MDMs within urine that offer high specificity and sensitivity for detection of prostate cancers as well as selective identification of clinically significant disease states. Prospective comparison between urine MDMs and PSA blood testing is necessary to discern the differential clinical impact of each screening methodology.
Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal ...immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy.
Infectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor ...extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins.
We investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis.
Single nucleotide polymorphisms in Mannosidase-1-α-2 (
) were significantly associated with BA and with other polymorphisms known to affect
expression but were not differentially enriched in either BA subtype. In zebrafish embryos,
knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer's vesicle, cardiac and liver heterotaxy, and dysregulated
and other developmental genes. Suboptimal
knockdown synergized with suboptimal EGFR signaling or suboptimal knockdown of the EGFR pathway gene, adenosine-ribosylation-factor-6, which had minimal effects individually, to reproduce biliary defects but not heterotaxy. In cultured mouse airway epithelium,
knockdown arrested ciliary development and motility.
mice, which experience respiratory failure, also demonstrated portal and bile ductular inflammation. Human BA liver and
liver exhibited reduced
expression and dysregulated ciliary genes, known to cause multisystem human laterality defects.
BA requiring transplantation associates with sequence variants in
.
regulates laterality, in addition to hepatobiliary morphogenesis, by regulating ciliogenesis in zebrafish and mice, providing a novel developmental basis for multisystem defects in BA.
► Introduce deuterated alkane ladder for retention time alignment. ► Automated feature selection for chemometric modelling of raw GC/MS data. ► Compare deuterated ladder with conventional ...feature-based alignment. ► Application to successful detection of gasoline in simulated fire debris.
Direct chemometric interpretation of raw chromatographic data (as opposed to integrated peak tables) has been shown to be advantageous in many circumstances. However, this approach presents two significant challenges: data alignment and feature selection. In order to interpret the data, the time axes must be precisely aligned so that the signal from each analyte is recorded at the same coordinates in the data matrix for each and every analyzed sample. Several alignment approaches exist in the literature and they work well when the samples being aligned are reasonably similar. In cases where the background matrix for a series of samples to be modeled is highly variable, the performance of these approaches suffers. Considering the challenge of feature selection, when the raw data are used each signal at each time is viewed as an individual, independent variable; with the data rates of modern chromatographic systems, this generates hundreds of thousands of candidate variables, or tens of millions of candidate variables if multivariate detectors such as mass spectrometers are utilized. Consequently, an automated approach to identify and select appropriate variables for inclusion in a model is desirable. In this research we present an alignment approach that relies on a series of deuterated alkanes which act as retention anchors for an alignment signal, and couple this with an automated feature selection routine based on our novel cluster resolution metric for the construction of a chemometric model. The model system that we use to demonstrate these approaches is a series of simulated arson debris samples analyzed by passive headspace extraction, GC–MS, and interpreted using partial least squares discriminant analysis (PLS-DA).
For the first time, the American (NASA) and Russian (ROSCOSMOS) space radiation transport codes, HZETRN and SHIELD respectively, are directly compared to each other. Calculations are presented for ...Galactic Cosmic Ray (GCR) minimum Hydrogen, Oxygen and Iron projectiles incident on a uniform Aluminum cylinder of varying thickness. Comparisons are made for the flux spectra of neutrons, light ions (Z≤ 2), heavy ions (Z> 2) and pions emitted from the back of the Aluminum cylinder. In order to provide more benchmark comparisons, some calculations with the GEANT and FLUKA transport codes are also shown.
Comparing HZETRN & SHIELD Transport Codes Norbury, John W.; Slaba, Tony C.; Sobolevsky, Nikolai ...
Life sciences in space research,
04/2017, Letnik:
14
Journal Article
For the first time, the American (NASA) and Russian (ROSCOSMOS) space radiation transport codes, HZETRN and SHIELD respectively, are directly compared to each other. Calculations are presented for ...Galactic Cosmic Ray (GCR) minimum Hydrogen, Oxygen and Iron projectiles incident on a uniform Aluminum cylinder of varying thickness. Comparisons are made for the flux spectra of neutrons, light ions, heavy ions and pions emitted from the back of the Aluminum cylinder. In order to provide more benchmark comparisons, some calculations with the GEANT and FLUKA transport codes are also shown.
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