Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment

Kisspeptin is a 54-amino acid peptide, encoded by the anti-metastasis gene KiSS-1, that activates G protein-coupled receptor 54 (GPR54). The kisspeptin-GPR54 system is critical to normal reproductive development. KiSS-1 gene expression is increased in the human placenta in normal and molar pregnancies. Circulating kisspeptin is dramatically increased in normal pregnancy, but levels in GTN have not previously been reported. The present study was designed to determine whether plasma kisspeptin levels are altered in patients with malignant GTN. Thirty-nine blood samples were taken from 11 patients with malignant GTN at presentation during and after chemotherapy. Blood was also sampled from nonpregnant and pregnant volunteers. Plasma kisspeptin IR and hCG concentrations were measured. Plasma kisspeptin IR concentration in nonpregnant (n = 16) females was <2 pmol/l. Plasma kisspeptin IR in females was 803 +/- 125 pmol/l in the first trimester of pregnancy (n = 13), 2,483 +/- 302 pmol/l in the third trimester of pregnancy (n = 7), and <2 pmol/l on day 15 postpartum (n = 7). Plasma kisspeptin IR and hCG concentrations in patients with malignant GTN were elevated at presentation and fell during and after treatment with chemotherapy in each patient (mean plasma kisspeptin IR: prechemotherapy 1,363 +/- 1,076 pmol/l vs. post-chemotherapy <2 pmol/l, P < 0.0001; mean plasma hCG: prechemotherapy 227,191 +/- 152,354 U/l vs. postchemotherapy 2 U/l, P < 0.0001). Plasma kisspeptin IR strongly positively correlated with plasma hCG levels (r(2) = 0.99, P < 0.0001). Our results suggest that measurement of plasma kisspeptin IR may be a novel tumor marker in patients with malignant GTN.     

Role of phenylalanine B10 in plant nonsymbiotic hemoglobins

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.     

Offspring production with cryopreserved sperm from a live-bearing fish Xiphophorus maculatus and implications for female fecundity

Xiphophorus fishes are well-established models for biomedical research of spontaneous or induced tumors, and their use in research dates back to the 1930s. Currently, 58 well-pedigreed lines exist among 24 Xiphophorus species housed as live animals at the Xiphophorus Genetic Stock Center. The technique of sperm cryopreservation has been applied to preserve these valuable genetic resources, and production of offspring has been reported with cryopreserved sperm in two species (X. helleri and X. couchianus). The goal of this research was to establish protocols for sperm cryopreservation and artificial insemination that yield live young in X. maculatus, a widely used research species. The objectives were to: 1) collect basic biological characteristics of males, and quantify the sperm production yield after crushing of dissected testis; 2) cryopreserve sperm from X. maculatus by adapting as necessary the protocols for sperm cryopreservation of X. helleri and X. couchianus; 3) use cryopreserved sperm to inseminate virgin females of X maculatus and other species (X. helleri and X. couchianus), and 4) compare experimental trials over a 3-year period to identify opportunities for improving female fecundity. In total, 117 males were used in this study with a standard length of 2.5 ± 0.3 cm (mean ± SD), body weight of 0.474 ± 0.149 g, and dissected testis weight of 7.1 ± 3.7 mg. Calculation of sperm availability showed 5.9 ± 2.8 × 10(6) sperm cells per mg of testis weight. Offspring were produced from cryopreserved sperm. Male-to-male variation (1-70%) was observed in post-thaw motility despite little variation in motility before freezing (60-90%) or genetic variation (~100 generations of sib-mating). Comparisons of biological factors of males did not have significant correlations with the production of live young, and the influence of females on production of young was identified from the comparison of artificial insemination over 3 years. Overall, this study describes offspring production from cryopreserved sperm in a third species of Xiphophorus fishes, and identifies the opportunities for improving female fecundity which is essential for establishment of germplasm repositories for Xiphophorus fishes.     

The Dual Arrhenius and Michaelis–Menten kinetics model for decomposition of soil organic matter at hourly to seasonal time scales

Decomposition of soil carbon stocks is one of the largest potential biotic feedbacks to climate change. Models of decomposition of soil organic matter and of soil respiration rely on empirical functions that relate variation in temperature and soil water content to rates of microbial metabolism using soil‐C substrates. Here, we describe a unifying modeling framework to combine the effects of temperature, soil water content, and soluble substrate supply on decomposition of soluble soil‐C substrates using simple functions based on process concepts. The model's backbone is the Michaelis–Menten equation, which describes the relationship between reaction velocity and soluble organic‐C and O₂ substrate concentrations at an enzyme's reactive site, which are determined by diffusivity functions based on soil water content. Temperature sensitivity is simulated by allowing the maximum velocity of the reaction (V_max) to vary according to Arrhenius function. The Dual Arrhenius and Michaelis–Menten kinetics (DAMM) model core was able to predict effectively observations from of laboratory enzyme assays of β‐glucosidase and phenol‐oxidase across a range of substrate concentrations and incubation temperatures. The model also functioned as well or better than purely empirical models for simulating hourly and seasonal soil respiration data from a trenched plot in a deciduous forest at the Harvard Forest, in northeastern United States. The DAMM model demonstrates that enzymatic processes can be intrinsically temperature sensitive, but environmental constrains of substrate supply under soil moisture extremes can prevent that response to temperature from being observed. We discuss how DAMM could serve as a core module that is informed by other modules regarding microbial dynamics and supply of soluble‐C substrates from plant inputs and from desorption of physically stabilized soil‐C pools. Most importantly, it presents a way forward from purely empirical representation of temperature and moisture responses and integrates temperature‐sensitive enzymatic processes with constraints of substrate supply.     

Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides

Aims: CSA‐13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F‐127 could interfere with the toxicity of CSA‐13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa. Methods and Results: The addition of pluronic acid F‐127 slightly decreased the number of dead cells after exposure to CSA‐13. Pluronic acid F‐127 blocked the permeabilizing effect of CSA‐13 on the plasma membrane of HUVEC (uptake of ethidium bromide, release of lactate dehydrogenase) without modifying its toxic effect on their mitochondrial function (MTT test, uptake of tetramethyl rhodamine ethyl ester). Conclusion: Pluronic acid F‐127 decreased the toxicity of CSA‐13 against eukaryotic cells without completely protecting them from mitochondrial damage at high concentrations of the drug. Significance and Impact of the Study: This work establishes that studies on the toxic effects of synthetic antimicrobials on eukaryotic cells should not only focus on the permeability of the plasma membrane but also on the integrity of the mitochondria.     

Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design

A fragment-based screen against human immunodeficiency virus type 1 (HIV) integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF) binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.     

The Molecular Basis for Recognition of CD1d/α-Galactosylceramide by a Human Non-Vα24 T Cell Receptor

CD1d-mediated presentation of glycolipid antigens to T cells is capable of initiating powerful immune responses that can have a beneficial impact on many diseases. Molecular analyses have recently detailed the lipid antigen recognition strategies utilized by the invariant Vα24-Jα18 TCR rearrangements of iNKT cells, which comprise a subset of the human CD1d-restricted T cell population. In contrast, little is known about how lipid antigens are recognized by functionally distinct CD1d-restricted T cells bearing different TCRα chain rearrangements. Here we present crystallographic and biophysical analyses of α-galactosylceramide (α-GalCer) recognition by a human CD1d-restricted TCR that utilizes a Vα3.1-Jα18 rearrangement and displays a more restricted specificity for α-linked glycolipids than that of iNKT TCRs. Despite having sequence divergence in the CDR1α and CDR2α loops, this TCR employs a convergent recognition strategy to engage CD1d/αGalCer, with a binding affinity (∼2 µM) almost identical to that of an iNKT TCR used in this study. The CDR3α loop, similar in sequence to iNKT-TCRs, engages CD1d/αGalCer in a similar position as that seen with iNKT-TCRs, however fewer actual contacts are made. Instead, the CDR1α loop contributes important contacts to CD1d/αGalCer, with an emphasis on the 4′OH of the galactose headgroup. This is consistent with the inability of Vα24− T cells to respond to α-glucosylceramide, which differs from αGalCer in the position of the 4′OH. These data illustrate how fine specificity for a lipid containing α-linked galactose is achieved by a TCR structurally distinct from that of iNKT cells.     

DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.     

On the Interplay of Telomeres,Nevi and the Risk of Melanoma

The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.