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Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+), n = 111; tcdB(-), n= 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+), n = 47; tcdB(-), n= 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+), n = 25; tcdB(-), n= 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB+, n = 111; tcdB-, n = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB+, n = 47; tcdB-, n = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB+, n = 25; tcdB-, n = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA registered QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

Approximately 10% of active galactic nuclei exhibit relativistic jets, which are powered by the accretion of matter onto supermassive black holes. Although the measured width profiles of such jets on large scales agree with theories of magnetic collimation, the predicted structure on accretion disk scales at the jet launch point has not been detected. We report radio interferometry observations, at a wavelength of 1.3 millimeters, of the elliptical galaxy M87 that spatially resolve the base of the jet in this source. The derived size of 5.5 ± 0.4 Schwarzschild radii is significantly smaller than the innermost edge of a retrograde accretion disk, suggesting that the M87 jet is powered by an accretion disk in a prograde orbit around a spinning black hole.

Black hole physics: A new window on the Galactic Centre Using Very Long Baseline Interferometry (VLBI) at the relatively short radio wavelength of 1.3 mm, a new intrinsic size estimate has been obtained for Sagittarius A*, the supermassive black hole candidate at the centre of the Milky Way. The resulting lower limit on the size of Sgr A* is less than the predicted size of the event horizon of the presumed black hole, suggesting that Sgr A* emissions centre not on the black hole itself but on the surrounding accretion flow. VLBI observations of the Galactic Centre at around 1.3 mm, less influenced by interstellar scattering than those made at longer wavelengths, open a new window onto black-hole physics that will become even more sensitive as new VLBI stations are built. The cores of most large galaxies are thought to harbour super massive black holes. Sagittarius A*, the compact source of radio, infrared and x-ray emission at the centre of the Milky Way, is the closest example of this phenomenon. This paper reports observations that set a limit less than the expected apparent size of the event horizon of the presumed black hole, suggesting that the bulk of Sgr A* emission may not be centred on the black hole, but arises in the surrounding accretion flow. The cores of most galaxies are thought to harbour supermassive black holes, which power galactic nuclei by converting the gravitational energy of accreting matter into radiation 1 . Sagittarius A* (Sgr A*), the compact source of radio, infrared and X-ray emission at the centre of the Milky Way, is the closest example of this phenomenon, with an estimated black hole mass that is 4,000,000 times that of the Sun 2 , 3 . A long-standing astronomical goal is to resolve structures in the innermost accretion flow surrounding Sgr A*, where strong gravitational fields will distort the appearance of radiation emitted near the black hole. Radio observations at wavelengths of 3.5 mm and 7 mm have detected intrinsic structure in Sgr A*, but the spatial resolution of observations at these wavelengths is limited by interstellar scattering 4 , 5 , 6 , 7 . Here we report observations at a wavelength of 1.3 mm that set a size of microarcseconds on the intrinsic diameter of Sgr A*. This is less than the expected apparent size of the event horizon of the presumed black hole, suggesting that the bulk of Sgr A* emission may not be centred on the black hole, but arises in the surrounding accretion flow.

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N -glycosylated at Asn 105 . The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105–122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal ‘tethered’ counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.

Malignant mesothelioma is a rare, highly aggressive cancer arising from mesothelial cells that line the pleural cavities. Approximately 80% of mesothelioma cases can be directly attributed to asbestos exposure. Additional suspected causes or co-carcinogens include other mineral fibres, simian virus 40 (SV40) and radiation. A mesothelioma epidemic in Turkey has demonstrated a probable genetic predisposition to mineral fibre carcinogenesis and studies of human tissues and animal models of mesothelioma have demonstrated genetic and epigenetic events that contribute to the multistep process of mineral fibre carcinogenesis. Several growth factors and their receptors have a significant role in the oncogenesis, progression and resistance to therapy of mesothelioma. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) have been shown as targets for therapy based on promising preclinical data. However, clinical trials of tyrosine kinase inhibitors in mesothelioma have been disappointing. Bcl-XL is an important antiapoptotic member of the Bcl-2 family and is overexpressed in several solid tumours, including mesothelioma. Reduction of Bcl-XL expression in mesothelioma induces apoptosis and engenders sensitisation to cytotoxic chemotherapeutic agents. Pharmacological inhibitors of antiapoptotic Bcl-2 family members continue to undergo refinement and have shown promise in mesothelioma.

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10−7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7–58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

We investigated whether differences in postoperative survival exist based on the presence and site of residual tumor (esophagus vs regional lymph nodes) after preoperative chemoXRT in patients with esophageal cancer. Based on these data, we reevaluated the role of EUS in identifying patients who maximally benefit from surgical esophageal resection after preoperative chemoXRT. We studied 97 consecutive esophageal cancer patients treated with preoperative chemoXRT and a potentially curative surgical procedure between 1998 to 2001. All patients had EUS examination prior to chemoXRT and 53 had a repeat EUS examination after chemoXRT but prior to surgery. Surgical resection specimens were analyzed for absence or presence of residual tumor and its location. Patients with residual tumor in the esophagus (pathT1-3N0) and patients without residual tumor (pathT0N0) had similar cumulative survival (p= 0.92). Patients with residual cancer in lymph nodes showed a trend toward shorter cumulative survival compared to patients without residual tumor in lymph nodes (p= 0.086). The actuarial survival in pathN1 group was lower than pathN0 group at 1, 2, and 3 yr. Patients with significant residual lymphadenopathy detected by EUS after therapy had significantly worse postoperative survival compared to patients with no residual lymphadenopathy (p= 0.028). In eight patients, we found that reliable cytologic identification of residual malignancy was technically feasible by EUS-FNA after chemoradiation therapy. Following preoperative chemoXRT and surgery, patients with residual tumor in the regional lymph nodes have lower actuarial survival at 1, 2, and 3 yr after surgery, compared to patients with path CR or with residual tumor only in the esophagus. EUS and EUS-guided FNA can be helpful in identifying residual tumor in the lymph nodes after preoperative chemoXRT to select patients who benefit maximally from surgery.

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal ‘tethered’ counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.

Approximately 10% of active galactic nuclei exhibit relativistic jets, which are powered by accretion of matter onto super massive black holes. While the measured width profiles of such jets on large scales agree with theories of magnetic collimation, predicted structure on accretion disk scales at the jet launch point has not been detected. We report radio interferometry observations at 1.3mm wavelength of the elliptical galaxy M87 that spatially resolve the base of the jet in this source. The derived size of 5.5 +/- 0.4 Schwarzschild radii is significantly smaller than the innermost edge of a retrograde accretion disk, suggesting that the M87 jet is powered by an accretion disk in a prograde orbit around a spinning black hole.