Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Ghrelin immunohistochemistry of gastric adenocarcinoma and mucoepidermoid carcinoma of salivary gland

Ghrelin (G-HH) synthesized in several tissues including salivary and stomach glands stimulates appetite in humans by modulating neuropeptide Y neurons in the hypothalamic arcuate nucleus. Loss of appetite is one of the most important symptoms of stomach cancer. We conducted a study using immunohistochemistry to determine whether salivary glands and stomach cancer tissues produce ghrelin. We determined that negative ghrelin immunohistochemistry discriminates tumors from normal tissues and may therefore further our understanding of the clinically important problem of reduced food intake and anorexia in cancer patients. Radioimmunoassay analyses confirmed that cancer cells do not produce a G-HH peptide, whereas normal cells yield this peptide.     

Prenatal screening for hemoglobinopathies. I. A prospective regional trial.

Prenatal hemoglobinopathy screening was chosen as a model system for the study of patient receptivity to unsolicited genetic information. Providers of prenatal care in Rochester, NY, were offered free testing of all their prenatal patients and genetic counseling of women found positive. The 18,907 prenatal samples tested in a 5-year period represented 35.1% of the pregnancies in the Rochester metropolitan region. A hemoglobinopathy was found in 810 pregnancies (4.3%). Of the 21 different types of hemoglobinopathies detected, the most common were sickle cell trait (59%), hemoglobin C trait (19%), beta-thalassemia trait (11%), and hemoglobin E trait (5%). At the time of phlebotomy, 75% of the pregnancies were of less than 18 wk duration. Sixty-six percent of the pregnancies occurred in patients unaware of their diagnosis, and 80% occurred in patients unaware that they might be at risk for a child with a serious blood disorder. Of the 810 positive pregnancies, 551 (68%) occurred in patients who came for counseling. Of 453 women counseled during their first screened pregnancy, 390 (86%) said they wanted their partners tested and 254 (55%) had their partner tested. In the 77 pregnancies thus found to be at risk, the couple was too late for prenatal diagnosis in 12 cases, and the condition for which the fetus was at risk was too mild in 12 cases. Prenatal diagnosis was offered in the remaining 53 pregnancies and was accepted by 25 couples (47%). These results indicate that unselected patients in the primary care setting in this region, even though pregnant, are receptive to and utilize genetic information.     

Inhibition of cell proliferation and dihydrofolate reductase by liposomes containing methotrexate-dimyristoylphosphatidylethanolamine derivatives and by the glycerophosphorylethanolamine analogs

Liposomes, which were prepared with the three methotrexate (MTX)-dimyristoylphosphatidylethanolamine (DMPE) derivatives described in the preceding paper, were tested for their ability to block proliferation of mouse 3T3 and L1210 cells. Tritiated deoxyuridine incorporation into DNA could be completely inhibited by liposomes sensitized with MTX-DMPE I (MTX-gamma-DMPE). Under similar conditions, liposomes containing MTX-DMPE II (MTX-alpha-DMPE) and MTX-DMPE III (MTX-alpha, gamma-diDMPE) produced partial and no inhibition, respectively. These effects on cell growth were paralleled by the capacity of liposomes, prepared with each of the DMPE derivatives, to inhibit dihydrofolate reductase isolated from L1210 cells. Analogous experiments with the three corresponding glycerophosphorylethanolamine (glyceroPE) analogs also indicated that MTX-glyceroPE I was the most effective inhibitor of both cell proliferation and enzymatic activity. However, MTX-DMPE I sensitized liposomes apparently enter target cells as a consequence of phagocytosis, and not via the ubiquitous methotrexate transport system that is employed by MTX-glyceroPE I. For example, novel use of thiamine pyrophosphate showed that this compound had no influence on inhibition of cell proliferation due to liposomes, whereas thiamine pyrophosphate could completely antagonize the inhibitory effects of methotrexate and MTX-glyceroPE I. The results are discussed with reference to possible therapeutic advantages of these liposomes.     

Spatial variability and temporal trends in water‐use efficiency of European forests

The increasing carbon dioxide (CO₂) concentration in the atmosphere in combination with climatic changes throughout the last century are likely to have had a profound effect on the physiology of trees: altering the carbon and water fluxes passing through the stomatal pores. However, the magnitude and spatial patterns of such changes in natural forests remain highly uncertain. Here, stable carbon isotope ratios from a network of 35 tree‐ring sites located across Europe are investigated to determine the intrinsic water‐use efficiency (iWUE), the ratio of photosynthesis to stomatal conductance from 1901 to 2000. The results were compared with simulations of a dynamic vegetation model (LPX‐Bern 1.0) that integrates numerous ecosystem and land–atmosphere exchange processes in a theoretical framework. The spatial pattern of tree‐ring derived iWUE of the investigated coniferous and deciduous species and the model results agreed significantly with a clear south‐to‐north gradient, as well as a general increase in iWUE over the 20th century. The magnitude of the iWUE increase was not spatially uniform, with the strongest increase observed and modelled for temperate forests in Central Europe, a region where summer soil‐water availability decreased over the last century. We were able to demonstrate that the combined effects of increasing CO₂ and climate change leading to soil drying have resulted in an accelerated increase in iWUE. These findings will help to reduce uncertainties in the land surface schemes of global climate models, where vegetation–climate feedbacks are currently still poorly constrained by observational data.