Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

The Non-catalytic ``Cap Domain'' of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Despite highly conserved core catalytic domains, members of the metallophosphoesterase (MPE) superfamily perform diverse and crucial functions ranging from nucleotide and nucleic acid metabolism to phospholipid hydrolysis. Unique structural elements outside of the catalytic core called ``cap domains'' are thought to provide specialization to these enzymes; however, no directed study has been performed to substantiate this. The cap domain of Rv0805, an MPE from Mycobacterium tuberculosis, is located C-terminal to its catalytic domain and is dispensable for the catalytic activity of this enzyme in vitro. We show here that this C-terminal extension (CTE) mediates in vivo localization of the protein to the cell membrane and cell wall as well as modulates expression levels of Rv0805 in mycobacteria. We also demonstrate that Rv0805 interacts with the cell wall of mycobacteria, possibly with the mycolyl-arabinogalactan-peptidoglycan complex, by virtue of its C terminus, a hitherto unknown property of this MPE. Using a panel of mutant proteins, we identify interactions between active site residues of Rv0805 and the CTE that determine its association with the cell wall. Finally, we show that Rv0805 and a truncated mutant devoid of the CTE produce different phenotypic effects when expressed in mycobacteria. Our study thus provides a detailed dissection of the functions of the cap domain of an MPE and suggests that the repertoire of cellular functions of MPEs cannot be understood without exploring the modulatory effects of these subdomains.

Temporal and spacial control of RNA stability in the early embryo of Drosophila melanogaster

Early embryogenesis in metazoa is controlled by maternally synthesized products. Among these products, the mature egg is loaded with transcripts representing approximately two thirds of the genome. A subset of this maternal RNA pool is degraded prior to the transition to zygotic control of development. This transfer of control of development from maternal to zygotic products is referred to as the midblastula transition (or MBT). It is believed that the degradation of maternal transcripts is required to terminate maternal control of development and to allow zygotic control of development to begin. Until now this process of maternal transcript degradation and the subsequent timing of the MBT has been poorly understood. I have demonstrated that in the early embryo there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is triggered by egg activation, and is targeted to specific classes of mRNAs through cis-acting elements in the 3' untranslated region (UTR}. The second pathway is activated 2 hr after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT. In addition, some transcripts fail to degrade at select subcellular locations adding an element of spatial control to RNA degradation. The spatial control of RNA degradation is achieved by protecting, or masking, transcripts from the degradation machinery. The RNA degradation and protection events are regulated by distinct cis-elements in the 3' untranslated region (UTR). These results provide the first systematic dissection of this highly conserved process in development and demonstrate that RNA degradation is a novel mechanism used for both temporal and spatial control of development.

On the nutrition of Polyphemus pediculus (L.). [Translation from: Trudy Instituta Biologii Vnutrennykh Vodnany 12(15) 170-174, 1966.]

Many sides of the biology of Polyphemus pediculus are adequately studied at the present time. Most complicated is the question of its feeding. Dissection does not give an idea of the composition of the food, since Polyphemus strongly grinds its food with its mandibles. For clarification of the composition of the food of Polyphemus pediculus, the authors carried out in July and August 1962 a series of experiments with the application of radio-carbon methods in order to judge the degree of utilization of one or other foods by the quantity of C14, accumulated in the body of the crustacean after feeding. Particular attention in these experiments was given to the question of the possibility of the utilization by polyphemus of plant food - bacteria, algae and detritus.

Length-weight relationship of fishes from Yemen waters (Gulf of Aden and Red Sea)

The data for this study were gathered between 1993 and 1996 on board commercial trawlers from Somalia, China and Yemen and also from the research vessel Ibn Magid belonging to the Marine Science and Resources Research Centre, Aden, Republic of Yemen. Fish were identified using the FAO species identification literature. All fish were measured to the nearest mm (total length) and weighed to the nearest g. Sex was determined by dissection after the length and weight had been measured. The length-weight relationships were calculated using least-squares regression on log-transformed data and the parameters of the relationship of the form of W=aL super(b) are summarized. Maximum and minimum size of fish sampled are also given. Common names and recent changes in nomenclature were taken from ICLARM's FishBase.

Age determination manual of the Alaska Fisheries Science Center Age and Growth Program

The Age and Growth Program at the Alaska Fisheries Science Center is tasked with providing age data in order to improve the basic understanding of the ecology and fisheries dynamics of Alaskan fish species. The primary focus of the Age and Growth Program is to estimate ages from otoliths and other calcified structures for age-structured modeling of commercially exploited stocks; however, the program has recently expanded its interests to include numerous studies on topics ranging from age estimate validation to the growth and life history of non-target species. Because so many applications rely upon age data and particularly upon assurances as to their accuracy and precision, the Age and Growth Program has developed this practical guide to document the age determination of key groundfish species from Alaskan waters. The main objective of this manual is to describe techniques specific to the age determination of commercially and ecologically important species studied by the Age and Growth Program. The manual also provides general background information on otolith morphology, dissection, and preparation, as well as descriptions of methods used to measure precision and accuracy of age estimates. This manual is intended not only as a reference for age readers at the AFSC and other laboratories, but also to give insight into the quality of age estimates to scientists who routinely use such data.

Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

On farm trial of freshwater pearl culture

On farm preliminary trial of freshwater pearl culture was done through 20 entrepreneurs in Boilor and Sutiakhali villages of Mymensingh district during 2004. A group of 20 enthusiastic women were selected and trained on the art of mantle tissue dissection, operation for mantle tissue implantation and preparation of ponds for pearl culture. A total of 200 juvenile freshwater mussel, Lamellidens marginalis, were collected from the wild and were used for mantle issue operation. The operated mussels were then transferred to farmer's pond and were subjected to observational trial. Length and weight of each of the test mussels were recorded before hanging them at a depth of 40 cm in net bags (3 mussels/net bag) in ponds at the rate of 24,700 mussels/ha of pond area. Ponds were routinely fertilized with organic and inorganic fertilizers thorough out the mussel rearing period. Water temperature, pH, plankton density and soil organic matter were monitored fortnightly. Growth of pearl is yet to be monitored through sacrifice of the mussels but X-ray photography of a few mussels indicated the initiation of pearl formation in most of them.

Factors affecting leaf morphology: a case study of Ranunculus natans C. A. Mey. (Ranunculaceae) in the arid zone of northwest China

In order to examine the role of environmental factors affecting foliar morphology, we performed a case study of leaf morphological variation of Ranunculus natans found in the arid zone of northwest China. We found that foliar phenotypic variation differed significantly between populations. We described substantial positive correlations between altitude and leaf area (LA) as well as leaf perimeter (LP), and also between longitude and number of teeth, along with dissection index (DI). The pH, conductivity, and salinity of the environment caused a significant decrease in both LA and LP. Ranked in terms of their impacts on leaf morphology, the six selected factors were: altitude > pH > conductivity > salinity > longitude > latitude. We found that foliar morphological variations are functional responses to water-quantity factors (e.g., altitude and longitude at regional scales) and water-availability relation factors (e.g., pH, conductivity, and salinity at local scales), rather than to temperature-relation factors (latitude). Therefore, altitude and longitude, along with pH, conductivity, and salinity, are the main factors that significantly influence foliar morphology in the arid zone of China. We found that main factors played major roles in plant phenotypic plasticity in a complex ecosystem, although different combinations and interactions of environmental and geographical factors in each local environment may obscure the general trends in trait changes along environmental gradients.

Rapid patterning of 1-D collagenous topography as an ECM protein fibril platform for image cytometry.

Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among potential applications as culture platforms for drug screening.

渤海湾盆地前第三系构造演化与煤成气成藏研究

The disequilibrium between supply and demand the east part of North China accelerated natural gas exploration in Bohai bay basin. Exploration practice showed that coal-derived gas is important resource. In searching of big to middle scaled coal derived gas field, and realize successive gas supply, the paper carried out integrated study on structural evolution of Pre-Tertiary and evaluation of reservoir forming condition of coal-derived gas. Study work of the paper was based on the following condition: available achievement in this field at present, good understanding of multiphase of tectonic movement. Study work was focused on geological evolution, source rock evaluation and dissection key factors controlling reservoir forming. Based on analysis of seismic data, drilling data, tectonic style of Pre-Tertiary was subdivided, with different tectonic style representing different tectonic process. By means of state of the art, such as analysis of balanced cross section, and erosion restoration, the paper reestablished tectonic history and analyzed basin property during different tectonic phase. Dynamic mechanism for tectonic movement and influence of tectonic evolution on tectonic style were discussed. Study made it clear that tectonic movement is intensive since Mesozoic including 2 phase of compressional movement (at the end of Indo-China movement, and Yanshan movement), 2 phase of extensional movement (middle Yanshan movement, and Himalayan movement), 2 phase of strike slip movement, as well as 2 phase of reversal movement (early Yanshan movement, and early Himalayan movement). As a result, three tectonic provinces with different remnant of strata and different tectonic style took shape. Based on afore mentioned study, the paper pointed out that evolution of Bohai bay basin experienced the following steps: basin of rift valley type (Pt2+3)-craton basin at passive continental margin (∈1－2)-craton basin at active continental margin (∈3- O)-convergent craton basin (C-T1+2)-intracontinental basin (J＋K). Superposition of basins in different stage was discussed. Aimed at tectonic feature of multiple phases, the paper put forward concept model of superposition of tectonic unit, and analyzed its significance on reservoir forming. On basis of the difference among 3 tectonic movements in Mesozoic and Cenozoic, superposition of tectonic unit was classified into the following 3 categories and 6 types: continuous subsidence type (I), subsidence in Mesozoic and uplift for erosion in Cenozoic (II1), repeated subsidence and uplift in Mesozoic and subsidence in Cenozoic (II2), repeated subsidence and uplift in Mesozoic and uplift for erosion in Cenozoic (II3), uplift for erosion in Mesozoic and subsidence in Cenozoic (II4), and continuous uplift (III). Take the organic facies analysis as link, the paper established relationship between sedimentary environment and organic facies, as well as organic facies and organic matter abundance. Combined information of sedimentary environment and logging data, the paper estimated distribution of organic matter abundance. Combined with simulation of secondary hydrocarbon generation, dynamic mechanism of hydrocarbon generation, and thermal history, the paper made static and dynamic evaluation of effective source rock, i.e. Taiyuan formation and Shanxi formation. It is also pointed out that superposition of tectonic unit of type II2, type II4, and type I were the most favorable hydrocarbon generation units. Based on dissection of typical primary coal-derived gas reservoir, including reservoir forming condition and reservoir forming process, the paper pointed out key factors controlling reservoir forming for Carboniferous and Permian System: a. remnant thickness and source rock property were precondition; b. secondary hydrocarbon generation during Himalayan period was key factor; c. tectonic evolution history controlling thermal evolution of source rock was main factor that determine reservoir forming; d. inherited positive structural unit was favorable accumulation direction; e. fault activity and regional caprock determined hydrocarbon accumulation horizon. In the end, the paper established reservoir forming model for different superposition of tectonic units, and pointed out promising exploration belts with 11 of the first class, 5 of the second class and 6 of the third class.

The validity of ultrasound estimation of muscle volumes

Infantolino, B., Gales, D., Winter, S., Challis, J., The validity of ultrasound estimation of muscle volumes, Journal of applied biomechanics, ISSN 1065-8483, Vol. 23, N?. 3, 2007 , pags. 213-217 RAE2008

Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice

Background: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. Methods: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. Results: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. Conclusions: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.