Common alkylating antitumor drugs, such as temozolomide, trigger their cytotoxicity by methylating the O6-position of guanosine in DNA. However, the therapeutic effect of these drugs is dampened by ...elevated levels of the DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which directly reverses this alkylation. As a result, assessing MGMT levels in patient samples provides an important predictor of therapeutic response; however, current methods available to measure this protein are indirect, complex and slow. Here we describe the design and synthesis of fluorescent chemosensors that report directly on MGMT activity in a single step within minutes. The chemosensors incorporate a fluorophore and quencher pair, which become separated by the MGMT dealkylation reaction, yielding light-up responses of up to 55-fold, directly reflecting repair activity. Experiments show that the best-performing probe retains near-native activity at mid-nanomolar concentrations. A nuclease-protected probe, NR-1, was prepared and tested in tumor cell lysates, demonstrating an ability to evaluate relative levels of MGMT repair activity in twenty minutes. In addition, a probe was employed to evaluate inhibitors of MGMT, suggesting utility for discovering new inhibitors in a high-throughput manner. Probe designs such as that of NR-1 may prove valuable to clinicians in selection of patients for alkylating drug therapies and in assessing resistance that arises during treatment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aircrew (consisting of flight attendants, pilots, or flight engineers/navigators) are exposed to cosmic ionizing radiation (CIR) at flight altitude, which originates from solar activity and galactic ...sources. These exposures accumulate over time and are considerably higher for aircrew compared to the general population, and even higher compared to U.S. radiation workers. Many epidemiological studies on aircrew have observed higher rates of specific cancers compared to the general population. Despite high levels of CIR exposure and elevated rates of cancer in aircrew, a causal link between CIR and cancer has yet to be established. Many challenges still exist in effectively studying this relationship, not the least of which is evaluating CIR exposure separately from the constellation of factors that occur as part of the flight environment. This review concentrates on cancer incidence and mortality observed among aircrew in epidemiologic studies in relation to CIR exposure and limitation trends observed across the literature. The aim of this review is to provide an updated comprehensive summary of the literature that will support future research by identifying epidemiological challenges and highlighting existing increased cancer concerns in an occupation where CIR exposure is anticipated to increase in the future.
This prospective trial was designed to compare the performance characteristics of five different screening tests in parallel for the detection of advanced colonic neoplasia: CT colonography (CTC), ...colonoscopy (OC), flexible sigmoidoscopy (FS), faecal immunochemical stool testing (FIT) and faecal occult blood testing (FOBT).
Average risk adults provided stool specimens for FOBT and FIT, and underwent same-day low-dose 64-multidetector row CTC and OC using segmentally unblinded OC as the standard of reference. Sensitivities and specificities were calculated for each single test, and for combinations of FS and stool tests. CTC radiation exposure was measured, and patient comfort levels and preferences were assessed by questionnaire.
221 adenomas were detected in 307 subjects who completed CTC (mean radiation dose, 4.5 mSv) and OC; 269 patients provided stool samples for both FOBT and FIT. Sensitivities of OC, CTC, FS, FIT and FOBT for advanced colonic neoplasia were 100% (95% CI 88.4% to 100%), 96.7% (82.8% to 99.9%), 83.3% (95% CI 65.3% to 94.4%), 32% (95% CI 14.9% to 53.5) and 20% (95% CI 6.8% to 40.7%), respectively. Combination of FS with FOBT or FIT led to no relevant increase in sensitivity. 12 of 45 advanced adenomas were smaller than 10 mm. 46% of patients preferred CTC and 37% preferred OC (p<0.001).
High-resolution and low-dose CTC is feasible for colorectal cancer screening and reaches sensitivities comparable with OC for polyps >5 mm. For patients who refuse full bowel preparation and OC or CTC, FS should be preferred over stool tests. However, in cases where stool tests are performed, FIT should be recommended rather than FOBT.
Despite the current reliance on blood cultures (BCs), the diagnosis of bloodstream infections (BSIs) can be sped up using new technologies performed directly on positive BC bottles. Two methods (the ...MALDI BioTyper system and FilmArray blood culture identification BCID panel) are potentially applicable. In this study, we performed a large-scale clinical evaluation (1,585 microorganisms from 1,394 BSI episodes) on the combined use of the MALDI BioTyper and FilmArray BCID panel compared to a reference (culture-based) method. As a result, the causative organisms of 97.7% (1,362/1,394) of the BSIs were correctly identified by our MALDI BioTyper and FilmArray BCID-based algorithm. Specifically, 65 (5.3%) out of 1,223 monomicrobial BCs that provided incorrect or invalid identifications with the MALDI BioTyper were accurately detected by the FilmArray BCID panel; additionally, 153 (89.5%) out of 171 polymicrobial BCs achieved complete identification with the FilmArray BCID panel. Conversely, full use of the MALDI BioTyper would have resulted in the identification of only 1 causative organism in 97/171 (56.7%) of the polymicrobial cultures. By applying our diagnostic algorithm, the median time to identification was shortened (19.5 h versus 41.7 h with the reference method; P < 0.001), and the minimized use of the FilmArray BCID panel led to a significant cost savings. Twenty-six out of 31 microorganisms that could not be identified were species/genera not designed to be detected with the FilmArray BCID panel, indicating that subculture was not dispensable for a few of our BSI episodes. In summary, the fast and effective testing of BC bottles is realistically adoptable in the clinical microbiology laboratory workflow, although the usefulness of this testing for the management of BSIs remains to be established.
Purpose
To compare multidetector computed tomography (MDCT) and cone-beam computed tomography (CBCT) regarding radiation, resolution, image noise, and image quality.
Methods
CBCT and 256-MDCT were ...compared based on three scan protocols: Standard-dose (≈24 mGy), reduced-dose (≈9 mGy), and low-dose (≈4 mGy). MDCT images were acquired in standard- and high-resolution mode (HR-MDCT) and reconstructed using filtered back projection (FBP) and iterative reconstruction (IR). Spatial resolution in linepairs (lp) and objective image noise (OIN) were assessed using dedicated phantoms. Image quality was assessed in scans of 25 cadaver heads using a Likert scale.
Results
OIN was markedly higher in FBP-MDCT when compared to CBCT. IR lowered the OIN to comparable values in standard-mode MDCT only. CBCT provided a resolution of 13 lp/cm at standard-dose and 11 lp/cm at reduced-dose vs. 11 lp/cm and 10 lp/cm in HR-MDCT. Resolution of 10 lp/cm was observed for both devices using low-dose settings. Quality scores of MDCT and CBCT did not differ at standard-dose (CBCT, 3.4; MDCT, 3.3-3.5;
p
> 0.05). Using reduced- and low-dose protocols, CBCT was superior (reduced-dose, 3.2 vs. 2.8; low dose, 3.0 vs. 2.3;
p
< 0.001).
Conclusion
Using the low-dose protocol, the assessed CBCT provided better objective and subjective image quality and equality in resolution. Similar image quality, but better resolution using CBCT was observed at higher exposure settings.
Key Points
•
The assessed CBCT device provided better image quality at lower doses
.
•
Objective and subjective image quality were comparable using higher exposure settings
.
•
CBCT showed superior spatial resolution in standard-dose and reduced-dose settings
.
•
Modern noise-reducing tools are used in CBCT devices currently
.
•
MDCT should be preferred for assessment of soft-tissue injuries and oncologic imaging
.
A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to ...model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10¹³ sâ»Â¹ when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.
Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for ...cancer therapeutics
. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins
. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach
. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
Background:
The lack of molecular targets for triple negative breast cancer (TNBC) has limited treatment options and reduced survivorship. Identifying new molecular targets may help improve patient ...survival and decrease recurrence and metastasis. As DNA repair defects are prevalent in breast cancer, we evaluated the expression and repair capacities of DNA repair proteins in preclinical models.
Methods:
DNA repair capacity was analyzed in four TNBC cell lines, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), MDA-MB-468 (MDA-468), and HCC1806, using fluorescence multiplex host cell reactivation (FM-HCR) assays. Expression of DNA repair genes was analyzed with RNA-seq, and protein expression was evaluated with immunoblot. Responses to the combination of DNA damage response inhibitors and primary chemotherapy drugs doxorubicin or carboplatin were evaluated in the cell lines.
Results:
Defects in base excision and nucleotide excision repair were observed in preclinical TNBC models. Gene expression analysis showed a limited correlation between these defects. Loss in protein expression was a better indicator of these DNA repair defects. Over-expression of PARP1, XRCC1, RPA, DDB1, and ERCC1 was observed in TNBC preclinical models, and likely contributed to altered sensitivity to chemotherapy and DNA damage response (DDR) inhibitors. Improved cell killing was achieved when primary therapy was combined with DDR inhibitors for ATM, ATR, or CHK1.
Conclusion:
Base excision and nucleotide excision repair pathways may offer new molecular targets for TNBC. The functional status of DNA repair pathways should be considered when evaluating new therapies and may improve the targeting for primary and combination therapies with DDR inhibitors.
Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; ...however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na
S), a hydrogen sulfide (H
S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na
S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na
S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na
S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na
S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ
) was not altered by Na
S whereas the survival of LN-18 ρ
cells was compromised supports this conclusion. When cells were treated with Na
S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H
S being a radiosensitizer. Therefore, this study provides the first evidence that H
S donors could be used in GBM therapy to potentiate radiation-induced killing.
CYFRA 21-1 serves as biomarker in several epithelial malignancies. However, its role in pancreatic cancer (PC) has not yet been investigated.
Within a prospective single-centre study serial blood ...samples were collected from patients with confirmed advanced PC. Pre-treatment values and weekly measurements of CYFRA 21-1, carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (assessed by Elecsys 2010, Roche Diagnostics) during palliative first-line chemotherapy were obtained. Biomarker data were correlated with objective response (determined by RECIST) as well as time to progression (TTP) and overall survival (OS) using uni- and multivariate analyses.
Seventy-eight patients were included, 45% of these received treatment in prospective clinical trials. Median TTP was 3.9 months, median OS 7.7 months. Pre-treatment CYFRA 21-1 levels were significantly associated with performance status (P=0.0399) and stage of disease (P=0.0001). Marker values before chemotherapy and at the 2-month staging of all three markers were considered significant predictors for objective treatment response. Pre-treatment CYFRA 21-1 levels, as well as CA 19-9 values, could be applied to define subgroups (categorised by tertiles) with a different OS outcome (CYFRA: 14.8 vs 7.1 vs 4.8 months, CA 19-9: 14.2 vs 7.1 vs 5.2 months; P<0.0001). CYFRA 21-1 and CA 19-9 (both as categorised and as continuous variables) showed a highly significant correlation with TTP and OS at nearly all-time points assessed in univariate analysis. In multivariate analysis, only CYFRA 21-1 and performance status were independent predictors for OS.
CYFRA 21-1 may serve as a valuable tool for monitoring treatment response and assessing prognosis in advanced PC.
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