Monomeric site-specific nucleases for genome editing

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.     

How Frequently are Words used on Naming Tests used in Spoken Conversation?

Naming, or word-finding ability, is typically assessed using measures that require a patient to name a pictured object. Words used less frequently tend to be more difficult to find when speaking; thus word frequency can be used as a measure of item difficulty on such tests. However, frequency data for words on naming measures has either not been used in the creation of these tests or has been derived from data on how frequently words have been used in written materials. The present study determined how frequently words on the Boston Naming Test (BNT), the Neuropsychological Assessment Battery (NAB) naming subtest, the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) naming subtest, and auditory naming measures developed by Hamberger and Seidel (2003, Journal of the International Neuropsychological Society, 9, 479) and Brandt et al. (2010, The Clinical Neuropsychologist, 24, 1326) are used in spoken language. Items on the auditory naming measures had the highest mean frequency, and the BNT items 30–60 had the lowest mean frequency. Furthermore, item frequency on the full BNT, NAB naming forms 1 and 2, and RBANS forms A correlated with item number, indicating items increase in difficulty on these tests, with trends in the same direction found for RBANS form B and Hamberger and Seidel’s auditory naming measure. Finally, differences in mean word frequency between tests underscore how interpretation of change in naming ability based on different measures should be made with caution.     

Innervation of the feline eyelids

Abstract

The innervation of the eyelids is incompletely understood. This is a particular problem for those who wish to develop animal models of eyelid dysfunction in humans. Blepharospasm, for example, is a disease of uncontrolled eyelid spasm that is difficult to manage clinically because the aetiology is not understood. The anatomical literature on eyelid innervation is sparse and even conflicting. We attempted to study eyelid innervation, both sensory and motor*, with injection of horseradish peroxidase (HRP) into the superior eyelidinferior eyelidand bulbar conjunctiva. We used 13 anaesthetized weanling cats. Shape and structure of the facial nucleus varied along its rostrocaudal extent, but there was a clear demarcation of lateral and medial division. HRP-filled facial nucleus cells were ipsilateral to the injection site, and label appeared throughout the rostrocaudal length. All injection sites, including bulbar conjunctiva, labelled facial nucleus neurons located with overlapping distribution, predominantly in the dorsal part of the lateral division. Likewise, heavy labelling occurred throughout the entire ipsilateral cranial cervical ganglion and the trigeminal ganglion in all kittens. Injection of upper or lower eyelids caused some labelling in the second through the fourth cervical spinal ganglia. [Neurol Res 1992; 14. 000-000]     

Orthotopic forelimb allotransplantation in the rat model

In this report, we present a rat orthotopic forelimb allotransplantation model. Eight forelimbs were transplanted from Brown Norway rats to Lewis rats. Axillary vessels of transplant were used as the vascular pedicles, which were anastomosed to the external jugular vein and common carotid artery of the recipient rat. The ulnar, radial, and median nerves were also repaired. Among rats, a tapered dose of cyclosporine was administered in five rats. In other three rats, no immunosuppressive therapy was given. The viability and signs of rejection of transplanted forelimbs, sensation recovery, bone healing, and histology were assessed up to the 90^th postoperative day. All of rats but one survived surgery. All of transplanted forelimbs survived. In the rats treated with cyclosporine the transplanted forelimbs achieved long‐term survival with motion and sensation recovery. On 90^th day after surgery, bone healing was achieved. There was no sign of rejection in histology. In the rats without cyclosporine treatment, the transplanted forelimbs experienced tissue necrosis started from day 12 postoperatively. This experimental study showed the feasibility of orthotopic forelimb allotransplantation in the rat model. © 2015 Wiley Periodicals, Inc. Microsurgery 36:672–675, 2016.     

Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus

The immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge (P value<0.05), anti-Tat ELISPOT responses at 2 weeks post challenge (P value <0.05) and peak neutralizing antibody titers pre-challenge (P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS.     

Feasibility and Acceptability of Delivering a Postpartum Weight Loss Intervention via Facebook: A Pilot Study

Objective

To evaluate the feasibility and acceptability of a Facebook-delivered postpartum weight loss intervention.

Tumor necrosis factor plays a protective role in experimental murine cutaneous leishmaniasis

The ability of mice to resist infection with L. major correlated directly with the capacity of their LNC to produce TNF in response to in vitro parasite challenge. Blocking TNF in vivo by passively administering anti-TNF antibodies exacerbated the course of L. major infection, resulting in substantially larger cutaneous lesions and elevated numbers of parasites within those lesions. In addition, treatment of infected mice with exogenous rHuTNF afforded host protection as evidenced by smaller lesion size and decreased parasite counts. Taken together, these results suggest a central role for TNF in resistance to L. major.