The ABCs of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera and Ceratitis.

Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by nonhomologous end join repair of clustered regularly interspaced short palindromic repeats and the CRISPR associated protein 9 (Cas9) (commonly known as CRISPR/Cas9) has been demonstrated to be an efficient method for creating knock-out mutants and can be utilized to create germline mutations in Tephritidae. In this paper, we describe detailed methods to knockout the white gene in three tephritid species in the genera Anastrepha, Bactrocera and Ceratitis, including the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the variables in tephritid systems that directed method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.

(2-Fluorophenylimino)tri(1-pyrrolyl)phosphorane.

The P atom of the title compound, C(18)H(16)FN(4)P, has a slightly distorted tetrahedral geometry. The P--N bond lengths range from 1.671 (3) to 1.680 (3) A, while the P==N bond is 1.517 (3) A. The pyrrolyl groups are arranged around the P atom in a chiral propeller-like geometry.

A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase.

The Xenopus laevis South African frog oocyte is a well suited and widely used system for protein biochemistry and functional studies. So far, two methods are commonly in use for the expression of exogenous proteins in this system. Investigators have the choice between cytoplasmic injections of in vitro synthesized cRNA or nuclear injections of cDNA. Here, we describe a new method for ion channel expression in oocytes, which consists of a coinjection of T7-driven cDNA and T7-RNA polymerase directly into the cytoplasm. This technique uses very limited amounts of purified enzyme and is also applicable to SP6 polymerase. Commercially available polymerases can also conveniently substitute for self-purified enzymes. The technique can be used for electrophysiological and biochemical analysis. In particular, high level expressions have been achieved for potassium (Shaker B, Kv1.2 and Kv1.3) and sodium (P mu 1.2) channels, and we also demonstrate efficient metabolic labeling of the calcium channel auxiliary beta 3 subunit. The properties of the channels expressed by this technique are indistinguishable from those of the channels expressed by classical methods. Expression of multi-subunit proteins was also achieved illustrating that the technique can be used for structure-function analyses. Moreover, this novel expression technique avoids many drawbacks of the two former techniques. It clearly bypasses the costly and time-consuming step of cRNA synthesis in vitro, prevents delicate cRNA manipulation and is easier to perform and more reliable than nuclear injection. Finally, it does not affect cell survival rate. These data indicate that the T7-RNA polymerase expression technique could be widely used in the future for the expression of exogenous proteins in the Xenopus oocyte system.

Disulfide bridge reorganization induced by proline mutations in maurotoxin.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.

Effect of maurotoxin, a four disulfide-bridged toxin from the chactoid scorpion Scorpio maurus, on Shaker K+ channels.

Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.

The I-II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit.

The auxiliary beta subunit is essential for functional expression of high voltage-activated Ca2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of alpha1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the alpha1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of alpha1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The beta subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length alpha1 subunit facilitates the cell surface expression of the channel in the absence of beta subunit. Our data suggest that the beta subunit favors Ca2+ channel plasma membrane expression by inhibiting an expression brake contained in beta-binding alpha1 sequences.

The [beta]2a subunit is a molecular groom for the Ca2+ channel inactivation gate.

Ca(2+) channel inactivation is a key element in controlling the level of Ca(2+) entry through voltage-gated Ca(2+) channels. Interaction between the pore-forming alpha(1) subunit and the auxiliary beta subunit is known to be a strong modulator of voltage-dependent inactivation. Here, we demonstrate that an N-terminal membrane anchoring site (MAS) of the beta(2a) subunit strongly reduces alpha(1A) (Ca(V)2.1) Ca(2+) channel inactivation. This effect can be mimicked by the addition of a transmembrane segment to the N terminus of the beta(2a) subunit. Inhibition of inactivation by beta(2a) also requires a link between MAS and another important molecular determinant, the beta interaction domain (BID). Our data suggest that mobility of the Ca(2+) channel I-II loop is necessary for channel inactivation. Interaction of this loop with other identified intracellular channel domains may constitute the basis of voltage-dependent inactivation. We thus propose a conceptually novel mechanism for slowing of inactivation by the beta(2a) subunit, in which the immobilization of the channel inactivation gate occurs by means of MAS and BID.

A new beta subtype-specific interaction in alpha1A subunit controls P/Q-type Ca2+ channel activation.

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.

Huperzine A--a potent acetylcholinesterase inhibitor of use in the treatment of Alzheimer's disease.

Huperzine A, 9-amino-13-ethylidene-11-methyl-4-azatricyclo [7.3.1.0(3,8)]trideca-3(8),6,11-trien-5-one, C15H18N2O, Mr = 242.32, monoclinic, P2(1)/n, a = 8.8574 (6), b = 12.1833 (7), c = 12.4278 (7) A, beta = 99.956 (5) degrees, V = 1320.9 (1) A3, Z = 4, Dx = 1.22 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 5.75 cm-1, F(000) = 520, T = 296 K, RF = 6.30% for 1402 reflections with Fo greater than or equal to 5 sigma(Fo) and 183 parameters. The pyridone ring is planar and the stereochemistry of the C(11)--C(12) double bond is E.

Control of epidemic methicillin-resistant Staphylococcus aureus.

We controlled the spread of epidemic methicillin-resistant Staphylococcus aureus (MRSA) infection in an 884-bed veterans' facility by cohorting known active MRSA carriers and MRSA-infected patients on one nursing unit. Simultaneously, all previously-institutionalized transfers into the veterans' facility were screened with swab cultures for MRSA at the time of admission. All MRSA patients were maintained on contact (gown and glove) or strict isolation and treated aggressively with topical and enteral antibiotics with the assistance of the infectious disease consultant. The monthly incidence of new MRSA patients dropped from a maximum of 16 per month to three or less per month within six months of instituting these infection control measures. There were no further MRSA bacteremias after the establishment of the MRSA cohort in a single unit. Aggressive cohort management of known MRSA patients and screening of previously-institutionalized patients on admission for MRSA controlled epidemic MRSA in this large institution.