[Transperineal prostate biopsy guided by dynamic intracavitary three-dimensional contrast-enhanced tomographic ultrasound imaging].

OBJECTIVE: To investigate the application value of dynamic intracavitary three-dimensional contrast-enhanced tomographic transrectal ultrasound imaging (3D-CETRUS TUI) in transperineal prostate biopsy for patients with different levels of PSA. METHODS: This prospective study included 180 patients with escalated levels of PSA (≥4 µg/L) or abnormal results of digital rectal examination. According to the level of PSA, the patients were divided into groups A (PSA ≤10 µg/L, n = 104) and B (PSA >10 µg/L, n = 76), and all underwent intracavitary 3D-CETRUS TUI followed by transperineal prostate biopsy under the guidance of two-dimensional gray-scale ultrasound, which involved a standard 12-core transperineal systematic prostate puncture by intracavitary biplane probe and then a 2-core puncture in the abnormal region displayed by 3D-CETRUS TUI. With the pathological results as the gold standard, comparisons were made between the diagnostic value of 3D-CETRUS TUI-guided biopsy and that of 12-core systematic biopsy. RESULTS: Prostate cancer was diagnosed in 69 (38.33%) of the 180 cases and in the specimens from 631 (25.03%) of the 2 520 punctures. The positive rates of prostate cancer were 37.5% (39/104) and 57.9% (44/76) in groups A and B, respectively. 3D-CETRUS TUI-guided prostate biopsy achieved a higher positive rate than 12-core systematic biopsy in both of the groups, with statistically significant difference in group A (P < 0.05), but not in B (P > 0.05). CONCLUSIONS: For patients with a PSA level ≤10 µg/L, dynamic intracavitary 3D-CETRUS TUI-guided transperineal prostate biopsy is more efficient than 12-core systematic biopsy in detecting the positive lesion, while for those with a PSA level > 10 µg/L, it effectively reduces the number of punctures.

Structural insights into PDZ-mediated interaction of NHERF2 and LPA(2), a cellular event implicated in CFTR channel regulation.

The formation of CFTR-NHERF2-LPA2 macromolecular complex in airway epithelia regulates CFTR channel function and plays an important role in compartmentalized cAMP signaling. We previously have shown that disruption of the PDZ-mediated NHERF2-LPA2 interaction abolishes the LPA inhibitory effect and augments CFTR Cl(-) channel activity in vitro and in vivo. Here we report the first crystal structure of the NHERF2 PDZ1 domain in complex with the C-terminal LPA2 sequence. The structure reveals that the PDZ1-LPA2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four LPA2 residues contributing to specific interactions. Comparison of the PDZ1-LPA2 structure to the structure of PDZ1 in complex with a different peptide provides insights into the diverse nature of PDZ1 substrate recognition and suggests that the conformational flexibility in the ligand binding pocket is involved in determining the broad substrate specificity of PDZ1. In addition, the structure reveals a small surface pocket adjacent to the ligand-binding site, which may have therapeutic implications. This study provides an understanding of the structural basis for the PDZ-mediated NHERF2-LPA2 interaction that could prove valuable in selective drug design against CFTR-related human diseases.

Crystal structure of the NHERF1 PDZ2 domain in complex with the chemokine receptor CXCR2 reveals probable modes of PDZ2 dimerization.

The formation of CXCR2-NHERF1-PLCß2 macromolecular complex in neutrophils regulates CXCR2 signaling and plays a key role in neutrophil chemotaxis and transepithelial neutrophilic migration. However, NHERF1 by itself, with only two PDZ domains, has a limited capacity in scaffolding the multiprotein-complex formation. Here we report the crystal structure of the NHERF1 PDZ2 domain in complex with the C-terminal CXCR2 sequence. The structure reveals that the PDZ2-CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. The structure also reveals two probable modes of PDZ2 dimerization where the two canonical ligand-binding pockets are well separated and orientated in a unique parallel fashion. This study provides not only the structural basis for the PDZ-mediated NHERF1-CXCR2 interaction, but also an additional example of how PDZ domains may dimerize, which both could prove valuable in understanding NHERF1 complex-scaffolding function in neutrophils.

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation.

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Polypeptide Substrate Accessibility Hypothesis: Gain-of-Function R206H Mutation Allosterically Affects Activin Receptor-like Protein Kinase Activity.

Although structurally similar to type II counterparts, type I or activin receptor-like kinases (ALKs) are set apart by a metastable helix-loop-helix (HLH) element preceding the protein kinase domain that, according to a longstanding paradigm, serves passive albeit critical roles as an inhibitor-to-substrate-binding switch. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder activation of ACVR1/ALK2 leading to sporadic heterotopic bone deposition in patients presenting with fibrodysplasia ossificans progressiva, or FOP. To determine the protein structural-functional basis for the gain of function, R206H mutant, Q207D (aspartate-substituted caALK2) and HLH subdomain-truncated (208 Ntrunc) forms were compared to one another and the wild-type enzyme through in vitro kinase and protein-protein interaction analyses that were complemented by signaling read-out (p-Smad) in primary mouse embryonic fibroblasts and Drosophila S2 cells. Contrary to the paradigm, the HLH subdomain actively suppressed the phosphotransferase activity of the enzyme, even in the absence of FKBP12. Unexpectedly, perturbation of the HLH subdomain elevated kinase activity at a distance, i.e., allosterically, at the ATP-binding and polypeptide-interacting active site cleft. Accessibility to polypeptide substrate (BMP Smad C-terminal tails) due to allosterically altered conformations of type I active sites within heterohexameric cytoplasmic signaling complexes-assembled noncanonically by activin-type II receptors extracellularly-is hypothesized to produce a gain of function of the R206H mutant protein responsible for episodic heterotopic ossification in FOP.

[Feasibility study on measuring composition concentration in turbid media by response variable transform method].

The present paper primarily studied the feasibility of the measurement of composition concentration in turbid media using response variable transform (RVT) method. With intralipid as the subject, the simulation of Monte Carlo in conjunction with the IAD algorithm and the experiment of double-integrating-sphere system combined with the IAD algorithm were conducted to verify the effectiveness to extract the scattering information. Furthermore, for the feasibility of the scattering coefficient to predict the concentration of intralipid, the results showed that measuring the component concentration in turbid media by means of RVT method is effective and feasible, and the relative error to predict the concentration with the linear relationship between scattering coefficient and concentration of intralipid is less than 10%.

Hypoxia-selective allosteric destabilization of activin receptor-like kinases: A potential therapeutic avenue for prophylaxis of heterotopic ossification.

Heterotopic ossification (HO), the pathological extraskeletal formation of bone, can arise from blast injuries, severe burns, orthopedic procedures and gain-of-function mutations in a component of the bone morphogenetic protein (BMP) signaling pathway, the ACVR1/ALK2 receptor serine-threonine (protein) kinase, causative of Fibrodysplasia Ossificans Progressiva (FOP). All three ALKs (-2, -3, -6) that play roles in bone morphogenesis contribute to trauma-induced HO, hence are well-validated pharmacological targets. That said, development of inhibitors, typically competitors of ATP binding, is inherently difficult due to the conserved nature of the active site of the 500+ human protein kinases. Since these enzymes are regulated via inherent plasticity, pharmacological chaperone-like drugs binding to another (allosteric) site could hypothetically modulate kinase conformation and activity. To test for such a mechanism, a surface pocket of ALK2 kinase formed largely by a key allosteric substructure was targeted by supercomputer docking of drug-like compounds from a virtual library. Subsequently, the effects of docked hits were further screened in vitro with purified recombinant kinase protein. A family of compounds with terminal hydrogen-bonding acceptor groups was identified that significantly destabilized the protein, inhibiting activity. Destabilization was pH-dependent, putatively mediated by ionization of a histidine within the allosteric substructure with decreasing pH. In vivo, nonnative proteins are degraded by proteolysis in the proteasome complex, or cellular trashcan, allowing for the emergence of therapeutics that inhibit through degradation of over-active proteins implicated in the pathology of diseases and disorders. Because HO is triggered by soft-tissue trauma and ensuing hypoxia, dependency of ALK destabilization on hypoxic pH imparts selective efficacy on the allosteric inhibitors, providing potential for safe prophylactic use.

Structural insights into neutrophilic migration revealed by the crystal structure of the chemokine receptor CXCR2 in complex with the first PDZ domain of NHERF1.

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.

New conformational state of NHERF1-CXCR2 signaling complex captured by crystal lattice trapping.

NHERF1 is a PDZ adaptor protein that scaffolds the assembly of diverse signaling complexes and has been implicated in many cancers. However, little is known about the mechanism responsible for its scaffolding promiscuity or its ability to bind to multiple targets. Computational studies have indicated that PDZ promiscuity may be attributed to its conformational dynamics, but experimental evidence for this relationship remains very limited. Here we examine the conformational flexibility of the NHERF1 PDZ1 domain using crystal lattice trapping via solving PDZ1 structure of a new crystal form. The structure, together with prior PDZ1 structures of a different space group, reveals that 4 of 11 ligand-interacting residues undergo significant crystal packing-induced structural changes. Most of these residues correspond to the residues involved in allosteric transition when a peptide ligand binds. In addition, a subtle difference in ligand conformations causes the same peptide to bind in slightly different modes in different crystal forms. These findings indicate that substantial structural flexibility is present in the PDZ1 peptide-binding pocket, and the structural substate trapped in the present crystal form can be utilized to represent the conformational space accessible to the protein. Such knowledge will be critical for drug design against the NHERF1 PDZ1 domain, highlighting the continued need for experimentally determined PDZ1-ligand complexes.

Practice guidelines for ultrasound-guided percutaneous microwave ablation for hepatic malignancy.

Primary liver cancer and liver metastases are among the most frequent malignancies worldwide, with an increasing number of new cases and deaths every year. Traditional surgery is only suitable for a limited proportion of patients and imaging-guided percutaneous thermal ablation has achieved optimistic results for management of hepatic malignancy. This synopsis outlines the first clinical practice guidelines for ultrasound-guided percutaneous microwave ablation therapy for hepatic malignancy, which was created by a joint task force of the Society of Chinese Interventional Ultrasound. The guidelines aim at standardizing the microwave ablation procedure and therapeutic efficacy assessment, as well as proposing the criteria for the treatment candidates.

Hypoxia during pregnancy in rats leads to early morphological changes of atherosclerosis in adult offspring.

Exposure to an adverse intrauterine environment increases the risk of cardiovascular disease later in adult life. However, the time course relationship between prenatal hypoxia and the onset of atherosclerosis in offspring remains unknown. The purpose of this study is to evaluate the role of reduced fetal oxygen supply on early development of atherogenesis in the adult offspring and further assess its susceptibility to sex-, hyperlipidemia-, and postnatal hypoxemia-related differences. Based on a 4 x 2 full factorial design consisting of four factors of maternal hypoxia, sex, hyperlipidemia, and postnatal hypoxemia, characteristics of growth were determined, and histopathological observation and morphometric analysis of the thoracic aortas were performed in Sprague-Dawley rat offspring. Intrauterine growth restriction, altered body shape at birth, and accelerated postnatal weight gain occurred in the maternal hypoxia group but did not occur in the control group. In 16-mo-old maternal hypoxia offspring, the thoracic aortas exhibited lesions similar to early events in atherosclerosis that involved impaired endothelial cells, thickening and fibration of intimas, infiltration of inflammatory cells to the subendothelial space, and migration and proliferation of vascular smooth muscle cells to the intima. In contrast, no detectable pathological changes were observed in the offspring without maternal hypoxia exposure. Morphometric analysis further demonstrated that prenatal hypoxia caused a significant thickening of intima (P < 0.001) with a main effect of 5.5 mum, an approximately twofold increase compared with controls. In addition, there was a positive additive relationship between prenatal hypoxia and hyperlipidemia on the intimal thickness (P < 0.05). There were no other main effects or interaction among these four factors. In summary, our results indicate that maternal hypoxia during pregnancy leads to early pathological appearances of atherogenesis in adult offspring. This effect was enhanced with hyperlipemia but was unaffected by postnatal hypoxia or sex.