Description: The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.

Description: Shiga toxin-producing Escherichia coli (STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like stx and eae , have been well studied, little is known about the prevalence of the E. coli hemolysin genes ( hlyA , ehxA , e-hlyA , and sheA ) in association with these factors. Hemolysins are potential virulence factors, and ehxA and hlyA have been associated with human illness, but the significance of sheA is unknown. Hence, 435 E. coli strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated ehxA subtype patterns in E. coli isolates from clinical, animal, and food sources. While sheA and ehxA were widely distributed, e-hlyA and hlyA were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried stx and/or eae ( P 〈 0.0001). ehxA subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (〉94.2%), with subtypes A/D being eae -negative STECs and subtypes B, C, E, and F eae positive. Unexpectedly, ehxA subtype patterns differed significantly between isolates collected from different sources ( P 〈 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular ehxA subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.

Description: The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

Description: It has been proposed recently that ATP hydrolysis in ATPase enzymes proceeds via an initial intermediate in which the dissociated γ-phosphate of ATP is bound in the protein as a metaphosphate (PγO3−). A combined quantum/classical analysis of this dissociated nucleotide state inside myosin provides a quantitative understanding of how the enzyme stabilizes this unusual metaphosphate. Indeed, in vacuum, the energy of the ADP3−·PγO3−·Mg2+ complex is much higher than that of the undissociated ATP4−. The protein brings it to a surprisingly low value. Energy decomposition reveals how much each interaction in the protein stabilizes the metaphosphate state; backbone peptides of the P-loop contribute 50% of the stabilization energy, and the side chain of Lys-185+ contributes 25%. This can be explained by the fact that these groups make strong favorable interactions with the α- and β-phosphates, thus favoring the charge distribution of the metaphosphate state over that of the ATP state. Further stabilization (16%) is achieved by a hydrogen bond between the backbone C=O of Ser-237 (on loop Switch-1) and a water molecule perfectly positioned to attack the PγO3− in the subsequent hydrolysis step. The planar and singly negative PγO3− is a much better target for the subsequent nucleophilic attack by a negatively charged OH− than the tetrahedral and doubly negative PγO42− group of ATP. Therefore, we argue that the present mechanism of metaphosphate stabilization is common to the large family of nucleotide-hydrolyzing enzymes. Methodologically, this work presents a computational approach that allows us to obtain a truly quantitative conception of enzymatic strategy.

Description: Targeting cancer cells with vitamin B12 (cobalamin) is hampered by unwanted physiologic tissue uptake mediated by transcobalamin. Adhering to good manufacturing practice, we have developed a new 99m Tc-cobalamin derivative ( 99m Tc(CO) 3 -[(4-amido-butyl)-pyridin-2-yl-methyl-amino-acetato] cobalamin, 99m Tc-PAMA-cobalamin). The derivative shows no binding to transcobalamin but is recognized by haptocorrin, a protein present in the circulation and notably expressed in many tumor cells. In this prospective study, we investigated cancer-specific uptake of 99m Tc-PAMA-cobalamin in 10 patients with various metastatic tumors. Methods: Ten patients with biopsy-proven metastatic cancer were included. Dynamic imaging was started immediately after injection of 300–500 MBq of 99m Tc-PAMA-cobalamin, and whole-body scintigrams were obtained at 10, 30, 60, 120, and 240 min and after 24 h. The relative tumor activity using SPECT/CT over the tumor region after 4 h was measured in comparison to disease-free lung parenchyma. Patients 3–10 received between 20 and 1,000 μg of cobalamin intravenously before injection of 99m Tc-PAMA-cobalamin. The study population comprised 4 patients with adenocarcinomas of the lung, 3 with squamous cell carcinomas of the hypopharyngeal region, 1 with prostate adenocarcinoma, 1 with breast, and 1 with colon adenocarcinoma. Results: The median age of the study group was 61 ± 11 y. Six of 10 patients showed positive tumor uptake on 99m Tc-PAMA-cobalamin whole-body scintigraphy. The scan was positive in 1 patient with colon adenocarcinoma, in 3 of 4 lung adenocarcinomas, in 1 of 3 hypopharyngeal squamous cell carcinomas, and in 1 breast adenocarcinoma. Renal uptake was between 1% and 3% for the left kidney. Predosing with cobalamin increased the tumor uptake and improved blood-pool clearance. The best image quality was achieved with a predose of 20–100 ug of cold cobalamin. The mean patient dose was 2.7 ± 0.9 mSv/patient. Conclusion: To our knowledge, we report for the first time on 99m Tc-PAMA-cobalamin imaging in patients with metastatic cancer disease and show that tumor targeting is feasible.

Description: Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ~90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential.

Description: Cell-cell contact inhibition and the mechanical environment of cells have both been shown to regulate YAP nuclear localization to modulate cell proliferation. Changes in cellular contractility by genetic, pharmacological, and matrix stiffness perturbations regulate YAP nuclear localization. However, because contractility and F-actin organization are interconnected cytoskeletal properties, it remains unclear which of these distinctly regulates YAP localization. Here we show that in the absence of cell-cell contact, actomyosin contractility suppresses YAP phosphorylation at Ser112, however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. We find that actin cytoskeletal integrity is essential for YAP nuclear localization, and can override phosphoregulation or contractility-mediated regulation of YAP nuclear localization. This actin-mediated regulation is conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced cytoskeletal integrity leading to nuclear exclusion of both YAP and Ser(P)112-YAP. These data provide evidence for two actin-mediated pathways for YAP regulation; one in which actomyosin contractility regulates YAP phosphorylation, and a second that involves cytoskeletal integrity-mediated regulation of YAP nuclear localization independent of contractility. We suggest that in non-contact inhibited cells, this latter mechanism may be important in low stiffness regimes, such as may be encountered in physiological environments.

Description: The tumor proliferation marker, Ki-67 index, is a well-established prognostic marker in gastroenteropancreatic neuroendocrine neoplasms (NENs). Noninvasive molecular imaging allows whole-body metabolic characterization of metastatic disease. We investigated the prognostic impact of 18 F-FDG PET in inoperable multifocal disease. Methods: Retrospective, dual-center analysis was performed on 89 patients with histologically confirmed, inoperable metastatic gastroenteropancreatic NENs undergoing 18 F-FDG PET/CT within the staging routine. Metabolic (PET-based) grading was in accordance with the most prominent 18 F-FDG uptake (reference tumor lesion): mG1, tumor-to-liver ratio of maximum standardized uptake value ≤ 1.0; mG2, 1.0–2.3; mG3, 〉2.3. Other potential variables influencing overall survival, including age, tumor origin, performance status, tumor burden, plasma chromogranin A (≥600 μg/L), neuron-specific enolase (≥25 μg/L), and classic grading (Ki-67–based) underwent univariate (log-rank test) and multivariate analysis (Cox proportional hazards model), with a P value of less than 0.05 considered significant. Results: The median follow-up period was 38 mo (95% confidence interval [CI], 27–49 mo); median overall survival of the 89 patients left for multivariate analysis was 29 mo (95% CI, 21–37 mo). According to metabolic grading, 9 patients (10.2%) had mG1 tumors, 22 (25.0%) mG2, and 57 (64.8%) mG3. On multivariate analysis, markedly elevated plasma neuron-specific enolase ( P = 0.016; hazard ratio, 2.9; 95% CI, 1.2–7.0) and high metabolic grade ( P = 0.015; hazard ratio, 4.7; 95% CI, 1.2–7.0) were independent predictors of survival. Conclusion: This study demonstrated the feasibility of prognostic 3-grade stratification of metastatic gastroenteropancreatic NENs by whole-body molecular imaging using 18 F-FDG PET.

Description: This study provides the first comprehensive quantification of translocator protein (TSPO) binding using SPECT and 6-chloro-2-(4'- 123 I-iodophenyl)-3-( N,N- diethyl)-imidazo[1,2-a]pyridine-3-acetamide ( 123 I-CLINDE) in neurologic patients. 123 I-CLINDE is structurally related to well-known PET ligands such as 18 F-PBR111 and 18 F-DPA-714. Methods: Six patients with cerebral stroke and 4 patients with glioblastoma multiforme (GBM) underwent 150-min dynamic SPECT scans with arterial blood sampling. Four of the patients were rescanned. All patients were genotyped for the rs6971 polymorphism. Volumes of interest were delineated on the individual SPECT scans and the coregistered MR images. Compartmental and graphical models using arterial input or the cerebellum as a reference region were used to quantify 123 I-CLINDE binding. Results: Among the 6 models investigated, the 2-tissue-compartment model with arterial input described the time–activity data best. Time–stability analyses suggested that acquisition time should be at least 90 min. Intersubject variation in the cerebellar distribution volume ( V T ) was clearly related to the TSPO genotype. In the stroke patients the V T in the periinfarction zone, compared with V T in the ipsilateral cerebellum, ranged from 1.4 to 3.4, and in the GBM patients the V T in the tumor, compared with the V T in the cerebellum, ranged from 1.8 to 3.4. In areas of gadolinium extravasation, 123 I-CLINDE binding parameters were not significantly changed. Thus, 123 I-CLINDE binding does not appear to be importantly affected by blood–brain barrier disruption. Conclusion: As demonstrated within a group of stroke and GBM patients, 123 I-CLINDE SPECT can be used for quantitative assessment of TSPO expression in vivo. Because of the absence of a region devoid of TSPO, reference tissue models should be used with caution. The 2-tissue-compartment kinetic analysis of a 90-min dynamic scan with arterial blood sampling is recommended for the quantification of 123 I-CLINDE binding with SPECT.

Description: CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nm affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with μm affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA.