Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome

The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line mutations at a pre-specified locus.     

Improved delivery of broadly neutralizing antibodies by nanocapsules suppresses SHIV infection in the CNS of infant rhesus macaques

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIV_SF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.     

ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration

The cellular events that precede myelination in the peripheral nervous system require rapid and dynamic morphological changes in the Schwann cell. These events are thought to be mainly controlled by axonal signals. But how signals on the axons are coordinately organized and transduced to promote proliferation, migration, radial sorting, and myelination is unknown. We describe that the axonal signal neuregulin-1 (NRG1) controls Schwann cell migration via activation of the atypical Dock180-related guanine nucleotide exchange factor (GEF) Dock7 and subsequent activation of the Rho guanine triphosphatases (GTPases) Rac1 and Cdc42 and the downstream c-Jun N-terminal kinase. We show that the NRG1 receptor ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. Dock7 knockdown, or expression of Dock7 harboring the Tyr-1118-to-Phe mutation in Schwann cells, attenuates the effects of NRG1. Thus, Dock7 functions as an intracellular substrate for ErbB2 to promote Schwann cell migration. This provides an unanticipated mechanism through which ligand-dependent tyrosine phosphorylation can trigger the activation of Rho GTPase-GEFs of the Dock180 family.     

Ecological Correlates to Lemur Community Structure in Southeast Madagascar

The Fandriana-Marolambo forest corridor is one of the largest (ca. 250,000 ha) and least explored tracts of unprotected forest in southeast Madagascar. Although published range maps show continuous distributions for many lemurs throughout the region, there are few data on lemur community structure in the corridor. We aimed to determine lemur community structure, with its ecological correlates (altitude, agriculture, selective logging, and hunting), in the Fandriana-Marolambo forest corridor. We surveyed 7 sites and sighted 4 nocturnal taxa (Avahi laniger, Cheirogaleus major, Lepilemur mustelinus, and Microcebus rufus) and 6 diurnal taxa (Eulemur rubriventer, E. fulvus rufus, E. f. fulvus, Propithecus diadema edwardsi, Hapalemur griseus griseus, and Varecia variegata variegata). Composition of the lemur community was broadly similar to that of nearby protected areas (Ranomafana and Mantadia National Parks). However, we sighted no Hapalemur aureus, H. simus, or Indri indri, and observed Propithecus diadema edwardsi and Varecia variegata variegata at only 1 site each. We sighted an apparent hybrid form of Eulemur fulvus fulvus and E. f. rufus that may represent a new hybrid zone for lemurs. After testing for spatial autocorrelation, lemur diversity correlates negatively with altitude and agricultural intensity. Though the Government of Madagascar is assessing the corridor as a new national park, we suggest conservation plans for local lemurs are complicated by population isolation and lack of data on minimum viable size of the proposed protected area.     

Selective measurement of NAPE-PLD activity via a PLA1/2-resistant fluorogenic N-acyl-phosphatidylethanolamine analog

N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase enzyme that converts NAPEs to bioactive N-acyl-ethanolamides. Altered NAPE-PLD activity may contribute to pathogenesis of obesity, diabetes, atherosclerosis, and neurological diseases. Selective measurement of NAPE-PLD activity is challenging, however, because of alternative phospholipase pathways for NAPE hydrolysis. Previous methods to measure NAPE-PLD activity involved addition of exogenous NAPE followed by TLC or LC/MS/MS, which are time and resource intensive. Recently, NAPE-PLD activity in cells has been assayed using the fluorogenic NAPE analogs PED-A1 and PED6, but these substrates also detect the activity of serine hydrolase-type lipases PLA₁ and PLA₂. To create a fluorescence assay that selectively measured cellular NAPE-PLD activity, we synthesized an analog of PED-A1 (flame-NAPE) where the sn-1 ester bond was replaced with an N-methyl amide to create resistance to PLA₁ hydrolysis. Recombinant NAPE-PLD produced fluorescence when incubated with either PED-A1 or flame-NAPE, whereas PLA₁ only produced fluorescence when incubated with PED-A1. Furthermore, fluorescence in HepG2 cells using PED-A1 could be partially blocked by either biothionol (a selective NAPE-PLD inhibitor) or tetrahydrolipstatin (an inhibitor of a broad spectrum of serine hydrolase-type lipases). In contrast, fluorescence assayed in HepG2 cells using flame-NAPE could only be blocked by biothionol. In multiple cell types, the phospholipase activity detected using flame-NAPE was significantly more sensitive to biothionol inhibition than that detected using PED-A1. Thus, using flame-NAPE to measure phospholipase activity provides a rapid and selective method to measure NAPE-PLD activity in cells and tissues.     

Questioning Ramayanas: A South Asian Tradition/Seeking Mahadevi: Constructing the Identities of the Hindu Great Goddess.

Questioning Ramayanas:. South Asian Tradition. Paula Richman. ed. Berkeley: University of California Press, 2001. 452 pp.
Seeking Mahadevi: Constructing the Identities of the Hindu Great Goddess. Tracy Pintchman. ed. Albany: State University of New York Press, 2001. 254 pp.     

Evidence of early butchery of giant lemurs in Madagascar

We report here definitive evidence of butchery, most probably associated with hunting, of giant extinct lemurs by early human settlers in Madagascar. Specimens of Palaeopropithecus ingens and Pachylemur insignis from two sites in southwestern Madagascar, Taolambiby and Tsirave, show classic signs of butchering. We compared these to the bones (also from Taolambiby) of butchered Propithecus verreauxi, a lemur still living in the region. The characteristics of the tool-induced extinct-lemur bone alterations (sharp cuts and chop marks near joints, oblique cuts along the shafts, spiral fractures, and percussion striae) suggest skinning, disarticulation, and filleting. Conclusive evidence of megafaunal modification by humans in Madagascar was limited previously to a few hippo and elephant bird bones and one extinct aye-aye tooth. New evidence comes not from archaeological sites, but from specimens collected in the early 1900s, without stratigraphic records, at "subfossil" sites (i.e., sites renowned for their late Pleistocene or Holocene fossils, often lacking human artifacts). Whereas these are hardly the most ideal samples for analysis of this kind, careful scrutiny of the characteristics of the cut marks has allowed us to document butchery beyond any reasonable doubt. One bone with definitive cut marks has been dated to the very earliest part of the human period in Madagascar. Continued, careful research on the bones in subfossil collections is warranted.     

Two Early Cretaceous Fossils Document Transitional Stages in Alvarezsaurian Dinosaur Evolution

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