Diversity and vertical distribution of epiphytic macrolichens in lowland rain forest and lowland cloud forest of French Guiana

Recent work on bryophyte diversity in lowland forests of northern South America has suggested the existence of a new type of cloud forest, the “tropical lowland cloud forest” (LCF). LCF occurs in river valleys with high air humidity and radiation fog, and is rich in epiphytes. We explored the lichenological characteristics of putative LCF in a lowland area (200–400 m a.s.l.) near Saül, central French Guiana, using macrolichens (including large crustose species) as indicator taxa. We analyzed macrolichen diversity on 16 trees in two 1 ha plots, in LCF and in lowland rain forest without fog (LRF). Sampling efficiency was ca. 80% in both forest types. Canopies of both LRF and LCF were richer in lichen species than understory trunks. Species richness of macrolichens was rather similar in the two forest types but species composition was significantly different. Cyanolichen richness in LCF was ca. 2.5 times higher than in LRF; in contrast, LRF had 4 times more species of green-algal Parmeliaceae. Our study suggests that cyanolichens except for Coccocarpiaceae serve as indicators of LCF. We explain the detected diversity patterns by differences in water availability due to fog precipitation and higher humidity. This is indicated by the higher relative air humidity in the lowland cloud forest, which was >6% higher than in the rain forest.     

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Supplemental Feeding for Ecotourism Reverses Diel Activity and Alters Movement Patterns and Spatial Distribution of the Southern Stingray,Dasyatis americana

Southern stingrays, Dasyatis americana, have been provided supplemental food in ecotourism operations at Stingray City Sandbar (SCS), Grand Cayman since 1986, with this site becoming one of the world’s most famous and heavily visited marine wildlife interaction venues. Given expansion of marine wildlife interactive tourism worldwide, there are questions about the effects of such activities on the focal species and their ecosystems. We used a combination of acoustic telemetry and tag-recapture efforts to test the hypothesis that human-sourced supplemental feeding has altered stingray activity patterns and habitat use at SCS relative to wild animals at control sites. Secondarily, we also qualitatively estimated the population size of stingrays supporting this major ecotourism venue. Tag-recapture data indicated that a population of at least 164 stingrays, over 80% female, utilized the small area at SCS for prolonged periods of time. Examination of comparative movements of mature female stingrays at SCS and control sites revealed strong differences between the two groups: The fed animals demonstrated a notable inversion of diel activity, being constantly active during the day with little movement at night compared to the nocturnally active wild stingrays; The fed stingrays utilized significantly (p<0.05) smaller 24 hour activity spaces compared to wild conspecifics, staying in close proximity to the ecotourism site; Fed stingrays showed a high degree of overlap in their core activity spaces compared to wild stingrays which were largely solitary in the spaces utilized (72% vs. 3% overlap respectively). Supplemental feeding has strikingly altered movement behavior and spatial distribution of the stingrays, and generated an atypically high density of animals at SCS which could have downstream fitness costs for individuals and potentially broader ecosystem effects. These findings should help environmental managers plan mitigating measures for existing operations, and develop precautionary policies regarding proposed feeding sites.     

Dual-source mass spectrometer with MALDI-LIT-ESI configuration

A novel linear ion trap (LIT) mass spectrometer with dual matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) ionization sources has been built in the MALDI-LIT-ESI configuration. The design features two independent ion source/ion optical channels connected to opposite ends of a single mass analyzer. The instrument consists of a commercial MALDI-LIT instrument modified by the addition of a home-built vacuum manifold, ion optical system, control electronics, and programming necessary to couple an atmospheric pressure interface to the commercial instrument. In addition to the added ESI functionality, the capabilities of the system also include simultaneous dual-channel ion introduction and analysis and high-duty cycle electronic switching (<1 s) between ion channels. Analytical and ion chemical applications of the dual-source system are explored. One analytical application is the enhanced protein sequence coverage achieved when using both ESI and MALDI to examine a tryptic digest of a six-protein mixture. The differences in the efficiency with which peptides in a mixture are ionized by the two methods give improved sequence coverage when both are applied. Other analytical applications include the use of the ions from one source as intensity or mass standards for the analyte ions from the other. An ion chemistry application involves the use of energy-resolved tandem mass spectrometry (MS/MS) to seek evidence for the generation of isomeric ions from a particular compound using the two ionization methods. A high level of agreement was achieved between the MS/MS spectra recorded under a variety of conditions after ESI and MALDI ionization; this provides evidence of the reproducibility and internal consistency of data from the dual source instrument. However, each of the peptides examined generated identical populations of structures in the two ionization methods under our conditions which are interpreted as involving slow cooling into the most stable minimum on the potential energy surface.     

Evaluating elastic network models of crystalline biological molecules with temperature factors, correlated motions, and diffuse x-ray scattering

In this study, the variance-covariance matrix of protein motions is used to compare several elastic network models within the theoretical framework of x-ray scattering from crystals. A set of 33 ultra-high resolution structures is used to characterize the average scaling behavior of the vibrational density of states and make comparisons between experimental and theoretical temperature factors. Detailed investigations of the vibrational density of states, correlations, and predicted diffuse x-ray scatter are carried out for crystalline Staphylococcal nuclease; correlations and diffuse x-ray scatter are also compared to predictions from the translation, libration, screw model and a liquid-like dynamics model. We show that elastic network models developed to best predict temperature factors without regard for the crystal environment have relatively strong long-range interactions that yield very short-ranged atom-atom correlations. Further, we find that the low-frequency modes dominate the variance-covariance matrix only for those models with a physically reasonable vibrational density of states, and the fraction of modes required to converge the correlations is higher than that typically used for elastic network model studies. The practical implications are explored using computed diffuse x-ray scatter, which can be measured experimentally.     

HIV-1 p24-immunoglobulin fusion molecule: a new strategy for plant-based protein production

We describe the engineering of a human immunodeficiency virus-1 (HIV-1) p24-immunoglobulin A (IgA) antigen-antibody fusion molecule for therapeutic purposes and its enhancing effect on fused antigen expression in tobacco plants. Although many recombinant proteins have been expressed in transgenic plants as vaccine candidates, low levels of expression are a recurring problem. In this paper, using the HIV p24 core antigen as a model vaccine target, we describe a strategy for increasing the yield of a recombinant protein in plants. HIV p24 antigen was expressed as a genetic fusion with the alpha2 and alpha3 constant region sequences from human Ig alpha-chain and targeted to the endomembrane system. The expression of this fusion protein was detected at levels approximately 13-fold higher than HIV p24 expressed alone, and a difference in the behaviour of the two recombinant proteins during trafficking in the plant secretory pathway has been identified. Expressing the antigen within the context of alpha-chain Ig sequences resulted in the formation of homodimers and the antigen was correctly recognized by specific antibodies. Furthermore, the HIV p24 elicited T-cell and antibody responses in immunized mice. The use of Ig fusion partners is proposed as a generic platform technology for up-regulating the expression of antigens in plants, and may represent the first step in a strategy to design new vaccines with enhanced immunological properties.