Pregnancy alters the rate and extent of drug metabolism, but little is known about the underlying molecular mechanism. We have found that 17β-estradiol (E2) upregulates expression of the major drug-metabolizing enzyme CYP2B6 in primary human hepatocytes. Results from promoter reporter assays in HepG2 cells revealed that E2 activates constitutive androstane receptor (CAR) and enhances promoter activity of CYP2B6, for which high concentrations of E2 reached during pregnancy were required. E2 triggered nuclear translocation of CAR in primary rat hepatocytes that were transiently transfected with human CAR as well as in primary human hepatocytes, further confirming transactivation of CAR by E2. E2-activated estrogen receptor (ER) also enhanced CYP2B6 promoter activity. The DNA-binding domain of ER was not required for the induction of CYP2B6 promoter activity by E2, suggesting involvement of a non-classical mechanism of ER action. Results from deletion and mutation assays as well as electrophorectic mobility shift and supershift assays revealed that two AP-1 binding sites (−1782/−1776 and −1664/−1658 of CYP2B6) are critical for ER-mediated activation of the CYP2B6 promoter by E2. Concurrent activation of both ER and CAR by E2 enhanced CYP2B6 expression in a synergistic manner. Our data demonstrate that at high concentrations reached during pregnancy, E2 activates both CAR and ER that synergistically induce CYP2B6 expression. These results illustrate pharmacological activity of E2 that would likely become prominent during pregnancy.
Cytochrome P450 2B6; estradiol; nuclear receptors; pregnancy
Cholesterol is known to modulate the physical properties of cell membranes but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol binding activity of NHERF1 largely abrogates its dynamic colocalization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.
Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria) to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20–1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids) present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia (http://evpedia.info) might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.
nanocosmos; communicasomes; exosomes; microvesicles; outer membrane vesicles; membrane vesicles; web portals; phylogenetic analyses
Myocardial perfusion scans contribute up to 20% of the estimated annual collective radiation dose to the U.S. population. We estimated potential future cancer risk from these scans by age at exposure and current frequency of use in the U.S.
Methods and Results
Usage patterns were determined from national survey data, and radionuclide dosage was based on current guidelines. Cancer risk projection models were generated based on the National Research Council Biologic Effects of Ionizing Radiation VII report, under the assumption that risk has a linear relationship with radiation exposure even at low doses. The mean projected number of radiation-related incident cancers and 95% uncertainty intervals (UI) were estimated using Monte Carlo simulations. Estimated risks for a scan performed at age 50 years ranged from 2 cancers/10,000 scans (95%UI:1–15) for a positron emission tomography ammonia-13 test to 25 (95%UI:9–58) cancers/10,000 scans for a dual-isotope (thallium-201+technetium-99m) scan. Risks were 50% lower at age 70 years, but were similar for males and females. Combination of cancer risk estimates with data on frequency of use suggested that the 9.1 million myocardial perfusion scans performed annually in the U.S. could result in 7400 (95%UI:3300–13700) additional future cancers.
The lifetime cancer risk from a single myocardial perfusion scan is small, and should be balanced against likely benefit and appropriateness of the test. The estimates depend on a number of assumptions including life-expectancy. They apply directly to asymptomatic individuals with life-expectancies similar to the general population. For individuals with a symptomatic clinical profile, on whom such scans are typically performed, the risks will be lower because of shorter life-expectancy.
nuclear medicine; perfusion; radioisotopes; cancer risks; computed tomography
The purpose of this study was to estimate the ratio of cancers prevented to induced (benefit-risk ratio) for CT colonography screening every five years from age 50-80.
Materials and methods
Radiation-related cancer risk was estimated using risk projection models based on the National Research Council's BEIR VII committee's report and screening protocols from the American College of Radiology Imaging Network's National CT Colonography Trial. Uncertainty limits (UL) were estimated using Monte-Carlo simulation methods. Comparative modelling with three colorectal cancer microsimulation models was used to estimate the potential reduction in colorectal cancer cases and deaths.
The estimated mean effective dose per CT colonography screen was 8mSv for females and 7mSv for males. The estimated number of radiation-related cancers from CT colonography screening every 5 years from age 50-80 was 150 cases/100,000 individuals (95%UL:80-280) for males and females. The estimated number of colorectal cancers prevented by CT colonography every 5 years from age 50-80 ranged across the three microsimulation models from 3580 to 5190/100,000, yielding a benefit-risk ratio that varied from 24:1(95%UL=13:1-45:1) to 35:1(95%UL=19:1-65:1). The benefit-risk ratio for cancer deaths was even higher than the ratio for cancer cases. Inclusion of radiation-related cancer risks from CT scans following-up extracolonic findings did not materially alter the results.
Concerns have been raised about recommending CT colonography as a routine screening tool because of the potential harms, including the radiation risks. Based on these models the benefits from CT colonography screening every five years from age 50-80 clearly outweigh the radiation risks.
Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis.
colorectal cancer; microvesicles; exosomes; ectosomes; metastasis; biomarker; secretome; APEX; label-free quantitative proteomics; nanoparticle tracking analysis
Although CT scans are very useful clinically, potential cancer risks exist from associated ionising radiation, in particular for children who are more radiosensitive than adults. We aimed to assess the excess risk of leukaemia and brain tumours after CT scans in a cohort of children and young adults.
In our retrospective cohort study, we included patients without previous cancer diagnoses who were first examined with CT in National Health Service (NHS) centres in England, Wales, or Scotland (Great Britain) between 1985 and 2002, when they were younger than 22 years of age. We obtained data for cancer incidence, mortality, and loss to follow-up from the NHS Central Registry from Jan 1, 1985, to Dec 31, 2008. We estimated absorbed brain and red bone marrow doses per CT scan in mGy and assessed excess incidence of leukaemia and brain tumours cancer with Poisson relative risk models. To avoid inclusion of CT scans related to cancer diagnosis, follow-up for leukaemia began 2 years after the first CT and for brain tumours 5 years after the first CT.
During follow-up, 74 of 178 604 patients were diagnosed with leukaemia and 135 of 176 587 patients were diagnosed with brain tumours. We noted a positive association between radiation dose from CT scans and leukaemia (excess relative risk [ERR] per mGy 0·036, 95% CI 0·005–0·120; p=0·0097) and brain tumours (0·023, 0·010–0·049; p<0·0001). Compared with patients who received a dose of less than 5 mGy, the relative risk of leukaemia for patients who received a cumulative dose of at least 30 mGy (mean dose 51·13 mGy) was 3·18 (95% CI 1·46–6·94) and the relative risk of brain cancer for patients who received a cumulative dose of 50–74 mGy (mean dose 60·42 mGy) was 2·82 (1·33–6·03).
Use of CT scans in children to deliver cumulative doses of about 50 mGy might almost triple the risk of leukaemia and doses of about 60 mGy might triple the risk of brain cancer. Because these cancers are relatively rare, the cumulative absolute risks are small: in the 10 years after the first scan for patients younger than 10 years, one excess case of leukaemia and one excess case of brain tumour per 10 000 head CT scans is estimated to occur. Nevertheless, although clinical benefits should outweigh the small absolute risks, radiation doses from CT scans ought to be kept as low as possible and alternative procedures, which do not involve ionising radiation, should be considered if appropriate.
US National Cancer Institute and UK Department of Health.
Background. Although using computed tomography (CT) can be greatly beneficial, the associated relatively high radiation doses have led to growing concerns in relation to potential associations with risk of future cancer. Very little has been published regarding the trends of CT use in young people. Therefore, our objective was to assess temporal and other patterns in CT usage among patients aged under 22 years in Great Britain from 1993 to 2002.
Methods. Electronic data were obtained from the Radiology Information Systems of 81 hospital trusts within Great Britain. All included patients were aged under 22 years and examined using CT between 1993 and 2002, with accessible radiology records.
Results. The number of CT examinations doubled over the study period. While increases in numbers of recorded examinations were seen across all age groups, the greatest increases were in the older patients, most notably those aged 15–19 years of age. Sixty percent of CT examinations were of the head, with the percentages varying with calendar year and patient age.
Conclusions. In contrast to previous data from the North of England, the doubling of CT use was not accompanied by an increase in numbers of multiple examinations to the same individual.
Two inducible temperate bacteriophages ΦS9 and ΦS63 from Clostridium perfringens were sequenced and analyzed. Isometric heads and long non-contractile tails classify ΦS9 and ΦS63 in the Siphoviridae family, and their genomes consist of 39,457 bp (ΦS9) and 33,609 bp (ΦS63) linear dsDNA, respectively. ΦS63 has 3′-overlapping cohesive genome ends, whereas ΦS9 is the first Clostridium phage featuring an experimentally proven terminally redundant and circularly permuted genome. A total of 50 and 43 coding sequences were predicted for ΦS9 and ΦS63, respectively, organized into 6 distinct lifestyle-associated modules typical for temperate Siphoviruses. Putative functions could be assigned to 26 gene products of ΦS9, and to 25 of ΦS63. The ΦS9 attB attachment and insertion site is located in a non-coding region upstream of a putative phosphorylase gene. Interestingly, ΦS63 integrates into the 3′ part of sigK in C. perfringens, and represents the first functional skin-element-like phage described for this genus. With respect to possible effects of lysogeny, we did not obtain evidence that ΦS9 may influence sporulation of a lysogenized host. In contrast, interruption of sigK, a sporulation associated gene in various bacteria, by the ΦS63 prophage insertion is more likely to affect sporulation of its carrier.
Clostridium perfringens; prophage; attachment site; sporulation; skin-element
Preeclapsia (PE) is a severe disorder that occurs during pregnancy, leading to maternal and fetal morbidity and mortality. PE affects about 3-8% of all pregnancies. In this study, we conducted liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS) to analyze serum samples depleted of the six most abundant proteins from normal and PE-affected pregnancies to profile serum proteins. A total of 237 proteins were confidently identified with < 1% false discovery rate from the two groups of duplicate analysis. The expression levels of those identified proteins were compared semiquantitatively by spectral counting. To further validate the candidate proteins with a quantitative mass spectrometric method, selective reaction monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant women with severe PE (n = 8) or normal pregnant women (n = 5) was conducted. α2-HS-glycoprotein (AHSG), retinol binding protein 4 (RBP4) and α-1-microglobulin/bikunin (AMBP) and Insulin like growth factor binding protein, acid labile subunit (IGFBP-ALS) were confirmed to be differentially expressed in PE using SRM (P < 0.05). Among these proteins, AHSG was verified by ELISA and showed a statistically significant increase in PE samples when compared to controls.
α2HS-glycoprotein; biomarkers; pre-eclampsia; spectrometry, mass, electrospray ionization; proteomics; serum
CT colonography has emerged as a potential screening tool for colorectal cancer that could provide good efficacy combined with greater acceptability than optical colonosocopy and fecal occult blood testing. Some organizations have raised concerns about potential harms, including perforation rates, radiation dose and the risk of radiation-related cancer, and have not recommended that it be currently used as a screening tool in the general population. In this paper we review the current evidence for these potential harms for CT colonography, and compare them to potential harms from the alternatives including colonoscopy and double-contrast barium enema.
Computed tomography; colorectal cancer; screening; radiation; risk; perforation; colonography
Cronobacter sakazakii is an opportunistic pathogen that actively invades host eukaryotic cells. To identify invasion factors responsible for the intestinal translocation of C. sakazakii, we constructed for the first time outer membrane protein X (OmpX) and A (OmpA) deletion mutants using the lambda Red recombination system. The ompX and ompA deletion mutants showed significantly reduced invasion of human enterocyte-like epithelial Caco-2 and human intestinal epithelial INT-407 cells, and significantly fewer mutant cells were recovered from the livers and spleens of rat pups. Furthermore, compared with intact target cells, the invasion and initial association potentials of the mutants increased at a rate similar to that of the wild type in tight-junction-disrupted target cells, suggesting that OmpX and OmpA are involved in basolateral invasion by C. sakazakii. This is the first report of C. sakazakii virulence determinants that are essential for basolateral invasion and that may be critical for the virulence of C. sakazakii.
Multi-detector computed tomography (MDCT) has been proposed as a tool for routine screening for coronary artery calcification (CAC) in asymptomatic individuals. As proposed, such screening could involve tens of millions of individuals, but detailed estimates of radiation doses and potential risk of radiation-induced cancer are not currently available. We estimated organ-specific radiation doses and associated cancer risks from CAC screening with MDCT, according to age, frequency and scan protocol.
Radiation doses to adult patients were calculated from a range of available protocols using Monte Carlo radiation transport. Radiation risk models, derived using data from Japanese atomic bomb survivors and medically-exposed cohorts, were used to estimate the excess lifetime risk of radiation-induced cancer.
Radiation dose from a single CAC CT scan varied more than 10-fold (effective dose range=0.8 to 10.5 mSv) depending on the protocol. In general higher radiation doses were associated with higher x-ray tube current, higher tube potential, and spiral scanning with low pitch, and retrospective gating. The wide dose variation also resulted in wide variation in estimated radiation-induced cancer risk. Assuming screening every five years from age 45-75 for men and from age 55-75 for women, the estimated excess lifetime cancer risk using the median dose of 2.3 mSv (0.8-10.5 mSv) was 42 cases/100,000 for men (range 14-200) and 62 cases/100,000 for women (range 21-300).
These radiation risk estimates can be compared to potential benefits from screening, when such estimates are available. Doses and hence risks can be minimized by using optimized protocols.
Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.
lipids; lipid metabolism disorders; phospholipids; phosphatidylinositols; spectrometry, mass, electrospray ionization; spectrometry, mass, matrix-assisted laser desorption-ionization
While the etiology of most childhood cancers is largely unknown, epidemiologic studies have consistently found an association between exposure to medical radiation during pregnancy and risk of childhood cancer in offspring. The relation between early life diagnostic radiation exposure and occurrence of pediatric cancer risks is less clear. This review summarizes current and historical estimated doses for common diagnostic radiologic procedures as well as the epidemiologic literature on the role of maternal prenatal, children’s postnatal and parental preconception diagnostic radiologic procedures on subsequent risk of childhood malignancies Risk estimates are presented according to factors such as the year of birth of the child, trimester and medical indication for the procedure, and the number of films taken. The paper also discusses limitations of the methods employed in epidemiologic studies to assess pediatric cancer risks, the effects on clinical practice of the results reported from the epidemiologic studies, and clinical and public health policy implications of the findings. Gaps in understanding and additional research needs are identified. Important research priorities include nationwide surveys to estimate fetal and childhood radiation doses from common diagnostic procedures, and epidemiologic studies to quantify pediatric and lifetime cancer risks from prenatal and early childhood exposures to diagnostic radiography, computed tomography, and fluoroscopically-guided procedures.
ionizing radiation; pediatric neoplasms; cancer risks; diagnostic radiography; CT
What is known about radiation exposure to physicians who perform cardiac interventions is reviewed and various factors that affect their exposure are discussed. There are wide variations in the radiation dose (up to 1000-fold) per procedure. Despite extensive improvements in equipment and technology, there has been little or no reduction in dose over time. The wide variation and lack of reduction in operator doses strongly suggests that more attention must be paid to factors influencing the operator dose. Numerous patient, physician and shielding factors influence the operator dose to different degrees. Operators can change some of these factors immediately, at minimal or no cost, with a substantial reduction in dose and potential cancer risk.
To estimate the risk of radiation-induced lung cancer mortality from three annual low-dose lung CT screens before age 55 years and the mortality reduction from screening (i.e. the efficacy) needed to outweigh these risks for never and current-smokers. The risk of radiation-induced breast cancer was also estimated for women.
The Biological Effectiveness of Ionizing Radiation VII committee’s risk models were used to estimate radiation risk. Lung cancer mortality rates (based on the Bach model for current and the Cancer Prevention Study for never-smokers) were used to estimate the mortality reduction needed to outweigh this risk.
For never-smokers the estimated excess lifetime risk of radiation-induced lung cancer mortality from annual screening age 40-42 was 1/10,000 (90% credibility interval:0.4-3) for males and 3/10,000 (2-6) for females. For current-smokers the estimated risks were approximately 2-fold higher, with wider credibility intervals. Risks from screening age 30-32 or 50-52 years were of similar magnitude. The mortality reduction required to outweigh these risks was, for male never-smokers:125%(40%-300%) age 30-32 years, 70%(30%-190%) age 40-42 years and 25%(10%-70%) age 50-52 years, and for male current-smokers:70%(20%-120%) age 30-32 years, 10%(3%-20%) age 40-42 years and 2%(1%-4%) age 50-52 years. These figures were 2-3 times higher for females because of the higher radiation risks. The risk of radiation-induced breast cancer was in the range of 3-6 cases/10,000 females screened.
Before age 50 the mortality reduction from lung CT screening that is required to outweigh the radiation risk may be substantial, and in some cases unattainable (i.e.>100%).
Ubiquitination is an important post-translational modification involved in diverse biological processes. Therefore, genomewide representation of the ubiquitination system for a species is important.
SCUD is a web-based database for the ubiquitination system in Saccharomyces cerevisiae (Baker's yeast). We first searched for all the known enzymes involved in the ubiquitination process in yeast, including E1, E2, E3, and deubiquitination enzymes. Then, ubiquitinated substrates were collected by literature search. Especially, E3 and deubiquitination enzymes are classified into classes and subclasses by their shared domains and unique functions. As a result, 42 different E3 enzymes were grouped into corresponding classes and subclasses, and 940 ubiquitinated substrates including mutant substrates were identified. All the enzyme and substrate information are interconnected by hyperlinks, which makes it easy to view the enzyme-specific ubiquitination information.
This database aims to represent a comprehensive yeast ubiquitination system, and is easily expandable with the further experimental data. We expect that this database will be useful for the research on the ubiquitination systems of other higher organisms.
SCUD is accessible at
Enterobacter sakazakii is an opportunistic pathogen that causes systemic bacteremia and meningitis with high mortality, and powdered infant formula is a frequent source of this bacterium. However, the mechanisms that this organism uses to invade and translocate through the intestinal barrier are unknown. Using Caco-2 epithelial cells, we were able to demonstrate penetration of E. sakazakii and to determine invasion-associated properties. We found that E. sakazakii entry and invasion were dependent on the exposure time and multiplicity of infection and required bacterial de novo protein synthesis but was independent of cell polarity in the presence of tight junctions. Moreover, the presence of actin filaments and microtubule structures was required, and disruption of the tight junction significantly enhanced the initial association with Caco-2 cells and the efficiency of invasion, which provides a possible explanation for the preferential occurrence of this infection in babies and neonates. This is the first description of E. sakazakii invasion of host intestinal cells, and our findings suggest that this emerging pathogen employs a novel invasion mechanism for development of systemic infection.
Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.
The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.
An in vitro transposition system, developed to facilitate gene disruption in Deinococcus radiodurans R1, has been used to inactivate the gene designated dr1819 in uvrA-1+ and uvrA-1 backgrounds. dr1819 encodes a protein with homology to a UV DNA damage endonuclease expressed by Schizosaccharomyces pombe. Interruption of dr1819 greatly sensitizes the uvrA-1 strain but not the uvrA-1+ strain to UV light, indicating that the dr1819 gene product is a component in a DNA repair pathway that can compensate for the loss of nucleotide excision repair in this species. Clones of dr1819 will restore UV resistance to UVS78, a uvrA-1 uvsE strain, indicating that dr1819 and uvsE are the same locus.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of common solid tumors. Using recently developed MALDI matrices for lipid profiling, we evaluated whether direct tissue MALDI MS analysis on proteins and lipids may classify human breast cancer samples according to the intrinsic subtype.
Thirty-four pairs of frozen, resected breast cancer and adjacent normal tissue samples were analyzed using histology-directed, MALDI MS analysis. Sinapinic acid and 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument.
Protein and lipid profiles distinguish cancer from adjacent normal tissue samples with the median prediction accuracy of 94.1%. Luminal, HER2+, and triple-negative tumors demonstrated different protein and lipid profiles, as evidenced by permutation P values less than 0.01 for 0.632+ bootstrap cross-validated misclassification rates with all classifiers tested. Discriminatory proteins and lipids were useful for classifying tumors according to the intrinsic subtype with median prediction accuracies of 80.0-81.3% in random test sets.
Protein and lipid profiles accurately distinguish tumor from adjacent normal tissue and classify breast cancers according to the intrinsic subtype.
protein; lipid; breast cancer; MALDI