Crystal Structures of the Human G3BP1 NTF2-Like Domain Visualize FxFG Nup Repeat Specificity

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded form of the G3BP1 NTF2-like domain was solved in two crystal forms to resolutions of 1.6 and 3.3 Å in space groups P2₁2₁2₁ and P6₃22 based on two different constructs, residues 1–139 and 11–139, respectively. Crystal packing of the N-terminal residues against a symmetry related molecule in the P2₁2₁2₁ crystal form might indicate a novel ligand binding site that, however, remains to be validated. The crystal structures give insight into the nuclear transportation mechanisms of G3BP and provide a basis for future structure based drug design.     

Multimodal Assessment of Long-Term Memory Recall and Reinstatement in a Combined Cue and Context Fear Conditioning and Extinction Paradigm in Humans

Learning to predict danger via associative learning processes is critical for adaptive behaviour. After successful extinction, persisting fear memories often emerge as returning fear. Investigation of return of fear phenomena, e.g. reinstatement, have only recently began and to date, many critical questions with respect to reinstatement in human populations remain unresolved. Few studies have separated experimental phases in time even though increasing evidence shows that allowing for passage of time (and consolidation) between experimental phases has a major impact on the results. In addition, studies have relied on a single psychophysiological dimension only (SCRs/SCL or FPS) which hampers comparability between different studies that showed both differential or generalized return of fear following a reinstatement manipulation. In 93 participants, we used a multimodal approach (fear-potentiated startle, skin conductance responses, fear ratings to asses fear conditioning (day 1), extinction (day 2) as well as delayed memory recall and reinstatement (day 8) in a paradigm that probed contextual and cued fear intra-individually. Our findings show persistence of conditioning and extinction memory over time and demonstrate that reinstated fear responses were qualitatively different between dependent variables (subjective fear ratings, FPS, SCRs) as well as between cued and contextual CSs. While only the arousal-related measurement (SCRs) showed increasing reactions following reinstatement to the cued CSs, no evidence of reinstatement was observed for the subjective ratings and fear-related measurement (FPS). In contrast, for contextual CSs, reinstatement was evident as differential and generalized reinstatement in fear ratings as well as generally elevated physiological fear (FPS) and arousal (SCRs) related measurements to all contextual CSs (generalized non-differential reinstatement). Returning fear after reinstatement likely depends on a variety of variables (experimental design, dependent measurements) and more systematic investigations with respect to critical determinants of reinstatement in humans are required.     

Glucose-6-Phosphate Dehydrogenase Deficiency in Nigerian Children

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (561 males and 561 females) aged 1 month to 15 years. The mean age was 7.4±3.2 years. Children of Yoruba ethnicity made up the largest group (77.5%) followed by those Igbo descent (10.6%) and those of Igede (10.2%) and Tiv (1.8%) ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females). Yoruba children had a higher prevalence (16.9%) than Igede (10.5%), Igbo (10.1%) and Tiv (5.0%) children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p = 0.0500). The odds for Igede and Tiv children were not significantly different from Yoruba children (p = 0.7528 and 0.9789 respectively). Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p = 0.0351). In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection.     

A Non-Coding Genomic Duplication at the HMX1 Locus Is Associated with Crop Ears in Highland Cattle

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.     

Yu Ping Feng San,an Ancient Chinese Herbal Decoction Containing Astragali Radix,Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix,Regulates the Release of Cytokines in Murine Macrophages

Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1∶2∶1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-κB by enhancing IκBα degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.     

Localization of Microfibrillar-Associated Protein 4 (MFAP4) in Human Tissues: Clinical Evaluation of Serum MFAP4 and Its Association with Various Cardiovascular Conditions

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease.     

Interactions of Platinum and Ruthenium Coordination Complexes with Pancreatic Phospholipase A2 and Phospholipids Investigated by MALDI TOF Mass Spectrometry

Phospholipase A₂ is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA₂ activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA₂ inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA₂ and phospholipids, as well as the application of MALDI‐TOF mass spectrometry for screening PLA₂ inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.     

Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.