Cinnamon has a long history of medicinal use and continues to be valued for its therapeutic potential for improving metabolic disorders such as type 2 diabetes. In this study, a phytochemically-enhanced functional food ingredient that captures water soluble polyphenols from aqueous cinnamon extract (CE) onto a protein rich matrix was developed. CE and cinnamon polyphenol-enriched defatted soy flour (CDSF) were effective in acutely lowering fasting blood glucose levels in diet-induced obese hyperglycemic mice at 300 and 600 mg/kg, respectively. To determine mechanisms of action, rat hepatoma cells were treated with CE and eluates of CDSF at a range of 1–25 µg/ml. CE and eluates of CDSF demonstrated dose-dependent inhibition of hepatic glucose production with significant levels of inhibition at 25 µg/ml. Furthermore, CE decreased the gene expression of two major regulators of hepatic gluconeogenesis, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. The hypoglycemic and insulin-like effects of CE and CDSF may help to ameliorate type 2 diabetes conditions.
Cinnamomum burmannii; cinnamon; diabetes; fasting blood glucose; glucose production
The present study demonstrated that defatted soybean flour (DSF) can sorb polyphenols from blueberry and cranberry juices while separating them from sugars. Depending on DSF concentration and juice dilution, the concentration of blueberry anthocyanins and total polyphenols sorbed to DSF ranged from 2 – 22 mg/g and 10 – 95 mg/g, respectively while the concentration of anthocyanins and proanthocyanidins in cranberry polyphenol-enriched DSF ranged from 2.5 – 17 mg/g and 21 – 101 mg/g, respectively. Blueberry polyphenols present in one serving of fresh blueberries (73g) were delivered in just 1.4 g of blueberry polyphenol-enriched DSF. Similarly, one gram of cranberry polyphenol-enriched DSF delivered the amount of proanthocyanidins available in three 240 ml servings of cranberry juice cocktail. The concentration of blueberry anthocyanins and total polyphenols eluted from DSF remained constant after 22 weeks of incubation at 37°C, demonstrating the high stability of the polyphenol-DSF matrix. LC-MS analysis of eluates confirmed DSF retained major cranberry and blueberry polyphenols remained intact. Blueberry polyphenol-enriched DSF exhibited significant hypoglycemic activities in C57bl/6J mice, and cranberry polyphenol-enriched DSF showed anti-microbial and anti-UTI activities in vitro, confirming its efficacy. The described sorption process provides a means to create protein-rich food ingredients containing concentrated plant bioactives without excess sugars, fats and water that can be incorporated in a variety of scientifically validated functional foods and dietary supplements.
polyphenols; anthocyanins; proanthocyanidins; soybean flour; nutrition; diabetes; antibacterial
We investigated the relationship of circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) with tumor glucose metabolism as defined by 18F-fluorodeoxyglucose (FDG) uptake since both have been associated with patient prognosis.
Materials & Methods
We performed a retrospective screen of patients at four medical centers who underwent FDG PET-CT imaging and phlebotomy prior to a therapeutic intervention for NSCLC. We used an Epithelial Cell Adhesion Molecule (EpCAM) independent fluid biopsy based on cell morphology for CTC detection and enumeration (defined here as High Definition CTCs or “HD-CTCs”). We then correlated HD-CTCs with quantitative FDG uptake image data calibrated across centers in a cross-sectional analysis.
We assessed seventy-one NSCLC patients whose median tumor size was 2.8 cm (interquartile range, IQR, 2.0–3.6) and median maximum standardized uptake value (SUVmax) was 7.2 (IQR 3.7–15.5). More than 2 HD-CTCs were detected in 63% of patients, whether across all stages (45 of 71) or in stage I disease (27 of 43). HD-CTCs were weakly correlated with partial volume corrected tumor SUVmax (r = 0.27, p-value = 0.03) and not correlated with tumor diameter (r = 0.07; p-value = 0.60). For a given partial volume corrected SUVmax or tumor diameter there was a wide range of detected HD-CTCs in circulation for both early and late stage disease.
CTCs are detected frequently in early-stage NSCLC using a non-EpCAM mediated approach with a wide range noted for a given level of FDG uptake or tumor size. Integrating potentially complementary biomarkers like these with traditional patient data may eventually enhance our understanding of clinical, in vivo tumor biology in the early stages of this deadly disease.
Cancer is currently diagnosed and treated based on the results of a tissue biopsy of the primary tumor or a metastasis using invasive techniques such as surgical resection or needle biopsy. New technology for retrieving cancer cells from the circulation, developed in the last 5 years, has made it possible to obtain a ‘fluid biopsy’ from the bloodstream without the need for an invasive procedure. This technological development makes it possible to diagnose and manage cancer from a blood test rather than from a traditional biopsy. It also allows the repeated sampling of cancer cells from a patient, making it possible, in a practical manner, to interrogate the disease repeatedly in order to understand the mechanisms by which cancer cells evolve within a given individual. The ability to obtain cancer cells repeatedly also has the potential to substantially advance drug development by enabling early ex vivo validation of both targets and early-stage compounds, as well as creating new efficiencies in the drug development process during clinical trials.
cancer diagnosis; circulating tumor cell; fluid biopsy; prognostic indicator
Acute myocardial infarction (MI), which involves the rupture of existing atheromatous plaque, remains highly unpredictable despite recent advances in the diagnosis and treatment of coronary artery disease. Accordingly, a biomarker that can predict an impending MI is desperately needed. Here, we characterize circulating endothelial cells (CECs) using the first automated and clinically feasible CEC 3-channel fluorescence microscopy assay in 50 consecutive patients with ST-elevation myocardial infarction (STEMI) and 44 consecutive healthy controls. CEC counts were significantly elevated in MI cases versus controls with median numbers of 19 and 4 cells/ml respectively (p = 1.1 × 10−10). A receiver-operating characteristic (ROC) curve analysis demonstrated an area under the ROC curve of 0.95, suggesting near dichotomization of MI cases versus controls. We observed no correlation between CECs and typical markers of myocardial necrosis (ρ=0.02, CK-MB; ρ=−0.03, troponin). Morphologic analysis of the microscopy images of CECs revealed a 2.5-fold increase (P<0.0001) in cellular area and 2-fold increase (P<0.0001) in nuclear area of MI CECs versus healthy control, age-matched CECs, as well as CECs obtained from patients with preexisting peripheral vascular disease. The distribution of CEC images containing from 2 up to 10 nuclei demonstrates that MI patients are the only group to contain more than 3 nuclei/image, indicating that multi-cellular and multi-nuclear clusters are specific for acute MI. These data indicate that CECs may serve as promising biomarkers for the prediction of atherosclerotic plaque rupture events.
The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G-protein-coupled sphingosine 1-phosphate receptors. Here we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Access to the binding pocket is completely occluded by the N-terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data and modeling, provides a detailed view of the molecular recognition and hydrophobic volume triggering that activates S1P1 resulting in the modulation of immune and stromal cell responses.
Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast, and colorectal cancer, and recent data suggests a similar role in late stage non-small cell lung cancer (NSCLC). However, due to sensitivity constraints in current enrichment-based CTC detection technologies, there is little published data about CTC prevalence rates and morphologic heterogeneity in early stage NSCLC, or the correlation of CTCs with disease progression and their usability for clinical staging. We investigated CTC counts, morphology, and aggregation in early stage, locally advanced, and metastatic NSCLC patients by using a fluid phase biopsy approach that identifies CTCs without relying on surface receptor-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. HD-CTCs were analyzed in blood samples from 78 chemotherapy-naïve NSCLC patients. 73% of the total population had a positive HD-CTC count (> 0 CTC in 1 mL of blood) with a median of 4.4 HD-CTCs/mL (range 0–515.6) and a mean of 44.7 (±95.2) HD-CTCs/mL. No significant difference in the medians of HD-CTC counts was detected between stage IV (n=31, range 0–178.2), stage III (n=34, range 0–515.6) and stages I/II (n=13, range 0–442.3). Furthermore, HD-CTCs exhibited a uniformity in terms of molecular and physical characteristics such as fluorescent cytokeratin intensity, nuclear size, frequency of apoptosis and aggregate formation across the spectrum of staging.
Our results demonstrate that, despite stringent morphologic inclusion criteria for the definition of HD-CTCs, the HD-CTC assay shows high sensitivity in the detection and characterization of both early and late stage lung cancer CTCs. Larger studies are warranted to investigate the prognostic value of CTC profiling in early stage lung cancer. This finding has implications for the design of larger studies examining screening, therapy, and surveillance in lung cancer patients.
Hematologic spread of carcinoma results in incurable metastasis; yet, the basic characteristics and travel mechanisms of cancer cells in the bloodstream are unknown. We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) without using surface protein-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. This “HD-CTC” assay finds >5 HD-CTCs/mL of blood in 80% of patients with metastatic prostate cancer (n=20), in 70% of patients with metastatic breast cancer (n=30), in 50% of patients with metastatic pancreatic cancer (n=18), and in 0% of normal controls (n=15). Additionally, it finds HD-CTC clusters ranging from 2 HDCTCs to greater than 30 HD-CTCs in the majority of these cancer patients. This initial validation of an enrichment-free assay demonstrates our ability to identify significant numbers of HD-CTCs in a majority of patients with prostate, breast and pancreatic cancers.
circulating tumor cells; fluid biopsy; microtumor emboli; pancreatic cancer; prostate cancer; breast cancer
Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate, and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity, and nuclear to cytoplasmic (N/C) ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.
Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may have on metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung (NSCLC), 18 pancreatic, 15 prostate stage IV cancer patients, and 15 normal blood donors (NBD). Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic (N/C) ratio between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.
Sampling circulating tumor cells from peripheral blood is ideally accomplished using assays that detect high numbers of cells and preserve them for downstream characterization. We sought to evaluate a method using enrichment free fluorescent labeling of CTCs followed by automated digital microscopy in patients with non-small cell lung cancer. Twenty-eight patients with non-small cell lung cancer and hematogenously seeded metastasis were analyzed with multiple blood draws. We detected CTCs in 68% of analyzed samples and found a propensity for increased CTC detection as the disease progressed in individual patients. CTCs were present at a median concentration of 1.6 CTCs per milliliter of analyzed blood in the patient population. Higher numbers of detected CTCs were associated with an unfavorable prognosis.
Circulating Tumor Cells; Cancer; Metastasis; prognosis; rare cell
The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 Å resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical α4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.
ultrahigh resolution; SRP; Ffh; GTPase; X-ray crystallography
Circulating tumor cells (CTCs) are associated with survival of cancer patients. Several methods have been developed to detect circulating tumor cells. The number of CTCs in NSCLC is lower than in other solid tumors. To date, trials are ongoing for a better understanding of CTCs. Besides association with prognosis, CTCs can be used to assess the efficacy of treatment and they are important substrates for molecular profiling of the tumor..
CTC; circulating tumor cells; HD-CTC; CellSearch; ISET; NSCLC
Defatted soybean flour (DSF) can efficiently sorb, concentrate, and stabilize polyphenols, but not sugars, from Concord grape juice, to yield grape polyphenol-enriched DSF. Sorption of grape polyphenols to DSF particles was dependent on the ratio of DSF and grape juice concentrate used, but not time of mixing or pH. Depending on ratios of starting materials, 1 g of grape polyphenol-enriched DSF contained 1.6–10.4 mg of anthocyanins, 7.5–93.1 mg of proanthocyanidins, and 20.5–144.5 mg of total polyphenols. LC-MS analysis of grape juice samples before and after addition and removal of DSF and eluate from grape polyphenol-enriched DSF confirmed that a broad range of grape compounds were sorbed to the DSF matrix. Finally, grape polyphenol-enriched DSF was able to significantly lower blood glucose levels in hyperglycemic C57BL/6J mice. The data indicate that grape polyphenol-enriched DSF can provide a high-protein, low-sugar ingredient for delivery of concentrated grape polyphenolics.
Concord grape juice; defatted soy flour; anthocyanins; proanthocyanidins; polyphenols; hypoglycemic; diabetes
A stochastic Markov chain model for metastatic progression is developed for primary lung cancer based on a network construction of metastatic sites with dynamics modeled as an ensemble of random walkers on the network. We calculate a transition matrix, with entries (transition probabilities) interpreted as random variables, and use it to construct a circular bi-directional network of primary and metastatic locations based on postmortem tissue analysis of 3827 autopsies on untreated patients documenting all primary tumor locations and metastatic sites from this population. The resulting 50 potential metastatic sites are connected by directed edges with distributed weightings, where the site connections and weightings are obtained by calculating the entries of an ensemble of transition matrices so that the steady-state distribution obtained from the long-time limit of the Markov chain dynamical system corresponds to the ensemble metastatic distribution obtained from the autopsy data set. We condition our search for a transition matrix on an initial distribution of metastatic tumors obtained from the data set. Through an iterative numerical search procedure, we adjust the entries of a sequence of approximations until a transition matrix with the correct steady-state is found (up to a numerical threshold). Since this constrained linear optimization problem is underdetermined, we characterize the statistical variance of the ensemble of transition matrices calculated using the means and variances of their singular value distributions as a diagnostic tool. We interpret the ensemble averaged transition probabilities as (approximately) normally distributed random variables. The model allows us to simulate and quantify disease progression pathways and timescales of progression from the lung position to other sites and we highlight several key findings based on the model.
Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in E. coli forms insoluble inclusion bodies that are difficult to denature and to refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10–50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1’s methyltransferase activity.
CARM1; PRMT family; methyl transferase; HaloTag; HaloLink resin; affinity purification
Clinical studies have demonstrated that circulating tumor cells (CTCs) are present in the blood of cancer patients with known metastatic disease across the major types of epithelial malignancies. Recent studies have shown that the concentration of CTCs in the blood is prognostic of overall survival in breast, prostate, colorectal, and non-small cell lung cancer. This study characterizes CTCs identified using the high-definition (HD)-CTC assay in an ovarian cancer patient with stage IIIC disease. We characterized the physical properties of 31 HD-CTCs and 50 normal leukocytes from a single blood draw taken just prior to the initial debulking surgery. We utilized a non-interferometric quantitative phase microscopy technique using brightfield imagery to measure cellular dry mass. Next we used a quantitative differential interference contrast microscopy technique to measure cellular volume. These techniques were combined to determine cellular dry mass density. We found that HD-CTCs were more massive than leukocytes: 33.6 ± 3.2 pg (HD-CTC) compared to 18.7 ± 0.6 pg (leukocytes), p < 0.001; had greater volumes: 518.3 ± 24.5 fL (HD-CTC) compared to 230.9 ± 78.5 fL (leukocyte), p < 0.001; and possessed a decreased dry mass density with respect to leukocytes: 0.065 ± 0.006 pg/fL (HD-CTC) compared to 0.085 ± 0.004 pg/fL (leukocyte), p < 0.006. Quantification of HD-CTC dry mass content and volume provide key insights into the fluid dynamics of cancer, and may provide the rationale for strategies to isolate, monitor or target CTCs based on their physical properties. The parameters reported here can also be incorporated into blood cell flow models to better understand metastasis.
circulating tumor cell; ovarian cancer; differential interference contrast; quantitative phase microscopy; cellular mass; cellular volume; cellular density
Cancer metastasis, the leading cause of cancer-related deaths, is facilitated in part by the hematogenous transport of circulating tumor cells (CTCs) through the vasculature. Clinical studies have demonstrated that CTCs circulate in the blood of patients with metastatic disease across the major types of carcinomas, and that the number of CTCs in peripheral blood is correlated with overall survival in metastatic breast, colorectal, and prostate cancer. While the potential to monitor metastasis through CTC enumeration exists, the basic physical features of CTCs remain ill defined and moreover, the corresponding clinical utility of these physical parameters is unknown. To elucidate the basic physical features of CTCs we present a label-free imaging technique utilizing differential interference contrast (DIC) microscopy to measure cell volume and to quantify sub-cellular mass-density variations as well as the size of subcellular constituents from mass-density spatial correlations. DIC measurements were carried out on CTCs identified in a breast cancer patient using the high-definition (HD) CTC detection assay. We compared the biophysical features of HD-CTC to normal blood cell subpopulations including leukocytes, platelets (PLT), and red blood cells (RBCs). HD-CTCs were found to possess larger volumes, decreased mass-density fluctuations, and shorter-range spatial density correlations in comparison to leukocytes. Our results suggest that HD-CTCs exhibit biophysical signatures that might be used to potentially aid in their detection and to monitor responses to treatment in a label-free fashion. The biophysical parameters reported here can be incorporated into computational models of CTC-vascular interactions and in vitro flow models to better understand metastasis.
circulating tumor cells; breast cancer; differential interference contrast microscopy; cellular volume; cellular density
Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The G protein-coupled chemokine receptor, CXCR4, is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5–3.2 Å resolution. All structures reveal a consistent homodimer with an interface involving helices V and VI that may be involved in regulating signaling. The location and shape of the ligand binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12 and with the HIV-1 glycoprotein gp120.
An ethanolic extract of Artemisia dracunculus L. (PMI-5011) was shown to be hypoglycemic in animal models for Type 2 diabetes and contain at least 6 bioactive compounds responsible for its anti-diabetic properties. To evaluate the bioavailability of the active compounds, high fat dietary induced obese C57BL/6J male mice were gavaged with PMI-5011 at 500 mg/kg body weight, after 4 h of food restriction. Blood plasma samples (200 uL) were obtained after ingestion, and the concentrations of the active compound in the blood sera were measured by electrospray LC-MS and determined to be maximal 4–6 h after gavage. Formulations of the extract with bioenhancers/solubilizers were evaluated in vivo for hypoglycemic activity and their effect on the abundance of active compounds in blood sera. At doses of 50–500 mg/kg/day, the hypoglycemic activity of the extract was enhanced 3–5 fold with the bioenhancer Labrasol, making it comparable to the activity of the anti-diabetic drug metformin. When combined with Labrasol, one of the active compounds, 2′, 4′-dihydroxy-4-methoxydihydrochalcone, was at least as effective as metformin at doses of 200–300 mg/kg/day. Therefore, bioenhancing agents like Labrasol can be used with multi-component botanical therapeutics such as PMI-5011 to increase their efficacy and/or to reduce the effective dose.
Wild berries are integral dietary components for Alaska Native tribes and a rich source of polyphenolic metabolites that can ameliorate metabolic disorders such as obesity and diabetes. In this study, five species of wild Alaskan berries (Vaccinium ovalifolium, V. uliginosum, Rubus chamaemorus, R. spectabilis, and Empetrum nigrum) were screened for bioactivity through a community-participatory research method involving three geographically-distinct tribal communities. Compositional analysis by HPLC and LC-MS2 revealed substantial site-specific variation in anthocyanins (0.01-4.39 mg/g-FW) and proanthocyanidins (0.74-6.25 mg/g-FW), and identified A-type proanthocyanidin polymers. R. spectabilis increased expression levels of preadipocyte-factor-1 (182%), and proanthocyanidin-enriched fractions from other species reduced lipid accumulation in 3T3-L1 adipocytes. Selected extracts reduced serum glucose levels in C57bl/6j mice by up to 45%. Local observations provided robust insights into effects of climatic fluctuations on berry abundance and quality, and preliminary site-specific compositional and bioactivity differences were noted, suggesting the need to monitor this Alaska Native resource as climate shifts impact the region.
Anthocyanins; proanthocyanidins; Vaccinium ovalifolium; Vaccinium uliginosum; Rubus spectabilis; Rubus chamaemorus; Empetrum nigrum; Traditional Ecological Knowledge; pref-1; adipocytes; diabetes; obesity; metabolic syndrome
This report describes two cases of urinary stress incontinence secondary to mesh repair of large abdominal hernias. Both patients had never experienced urinary incontinence before their hernia repair. In both cases, polypropylene nets were inserted to stabilise the abdominal wall. Immediately after the intervention, both patients became stress incontinent. Probably pressure transmission to the pelvic floor was increased due to stabilisation of the abdominal wall, which worked as a “windpipe” before surgery. After the insertion of suburethral tapes the incontinence resolved.
Coactivator-associated arginine methyltransferase 1 (CARM1), the histone arginine methyltransferase and coactivator for many transcription factors, is subject to multiple post-translational modifications (PTMs). To unbiasedly investigate novel CARM1 PTMs we employed high-resolution top-down mass spectrometry. Surprisingly, mouse CARM1 expressed in insect and mammalian expression systems was completely dimethylated at a single site in the C-terminal domain (CTD). We demonstrate that dimethylation of CARM1 occurs both in vivo and in vitro and proceeds via an automethylation mechanism. To probe function of automethylation, we mutated arginine 551 to lysine to create an automethylation-deficient CARM1. Although mutation of CARM1's automethylation site did not affect its enzymatic activity, it did impair both CARM1-activated transcription and pre-mRNA splicing. These results strongly imply that automethylation of CARM1 provides a direct link to couple transcription and pre-mRNA splicing in a manner differing from the other steroid receptor coactivators. Furthermore, our study identifies a self-regulatory signaling mechanism from CARM1's catalytic domain to its CTD.
Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.
lipidic cubic phase; G protein-coupled receptor; minibeam; microcrystallography
Structural genomics discovery projects require ready access to both X-ray and NMR instrumentation which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive frequent use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large government-run synchrotron facilities for data collection. In an effort to eliminate the need to ship crystals for data collection, we have developed the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.