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The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs.

Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.

PURPOSE We recorded pattern electroretinograms (PERGs) simultaneously from each eye in mice using binocular stimulation and a common noncorneal electrode. METHODS The PERG was derived simultaneously from each eye in 71 ketamine/xylazine anesthetized mice (C57BL/6J, 4 months old) from subcutaneous needles (active, snout; reference, back of the head; ground, root of the tail) in response to contrast-reversal of gratings (0.05 cycles/deg, >95% contrast) generated on two custom-made light-emitting diode (LED) tablets alternating at slight different frequencies (OD, 0.984 Hz; OS, 0.992 Hz). Independent PERG signals from each eye were retrieved using one channel continuous acquisition and phase-locking average (OD, 369 epochs of 492 ms; OS, 372 epochs of 496 ms). The PERG was the average of three consecutive repetitions. RESULTS Binocular snout PERGs had high amplitude (mean, 25.3 μV, SD 6.6) and no measurable interocular cross-talk. Responses were reliable (test-retest variability within-session, 14%, SD 7; between sessions, 25%, SD 9; interocular asymmetry within-session, 9%, SD 7; between sessions, 13%, SD 5). Retinal ganglion cells (RGCs) were the main source of the binocular snout PERG, as optic nerve crush in three mice abolished the signal. CONCLUSIONS The PERG, a sensitive measure of RGC function, is used increasingly in mouse models of glaucoma and optic nerve disease. Compared to current methods, the binocular snout PERG represents a substantial improvement in terms of simplicity and speed. It also overcomes limitations of corneal electrodes that interfere with invasive procedures of the eye and facilitates experiments based on comparison between the responses of the two eyes.

IMPORTANCE We developed a novel strategy for treatment of Leber hereditary optic neuropathy (LHON) caused by a mutation in the nicotinamide adenine dinucleotide dehydrogenase subunit IV (ND4) mitochondrial gene. OBJECTIVE To demonstrate the safety and effects of the gene therapy vector to be used in a proposed gene therapy clinical trial. DESIGN AND SETTING In a series of laboratory experiments, we modified the mitochondrial ND4 subunit of complex I in the nuclear genetic code for import into mitochondria. The protein was targeted into the organelle by agency of a targeting sequence (allotopic expression). The gene was packaged into adeno-associated viral vectors and then vitreally injected into rodent, nonhuman primate, and ex vivo human eyes that underwent testing for expression and integration by immunohistochemical analysis and blue native polyacrylamide gel electrophoresis. During serial follow-up, the animal eyes underwent fundus photography, optical coherence tomography, and multifocal or pattern electroretinography. We tested for rescue of visual loss in rodent eyes also injected with a mutant G11778A ND4 homologue responsible for most cases of LHON. EXPOSURE Ocular infection with recombinant adeno-associated viral vectors containing a wild-type allotopic human ND4 gene. MAIN OUTCOMES AND MEASURES Expression of human ND4 and rescue of optic neuropathy induced by mutant human ND4. RESULTS We found human ND4 expressed in almost all mouse retinal ganglion cells by 1 week after injection and ND4 integrated into the mouse complex I. In rodent eyes also injected with a mutant allotopic ND4, wild-type allotopic ND4 prevented defective adenosine triphosphate synthesis, suppressed visual loss, reduced apoptosis of retinal ganglion cells, and prevented demise of axons in the optic nerve. Injection of ND4 in the ex vivo human eye resulted in expression in most retinal ganglion cells. Primates undergoing vitreal injection with the ND4 test article and followed up for 3 months had no serious adverse reactions. CONCLUSIONS AND RELEVANCE Expression of our allotopic ND4 vector in the ex vivo human eye, safety of the test article, rescue of the LHON mouse model, and the severe irreversible loss of visual function in LHON support clinical testing with mutated G11778A mitochondrial DNA in our patients.

Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. The MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here report the crystal structure of the inner membrane MtrD multidrug efflux pump, which reveals a novel structural feature that is not found in other RND efflux pumps.

The Rv1217c–Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c–Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.

PURPOSE. To develop a mouse model of inducible, chronic retinal ganglion cell (RGC) dysfunction not associated with cell death. METHODS. Eighteen C57BL/6J mice were longitudinally tested with pattern electroretinogram (PERG) and spectral-domain optical coherence tomography (OCT) before and after aspiration of the contralateral superior colliculus (SC), which removed terminals of optic tract axons and the superficial layers of the SC. At the 4-month end points, retinas were harvested for Brn3b immunostaining and BDNF immunoblotting. RESULTS. The PERG lost approximately 60% of its baseline amplitude (P < 0.01) within the first day after lesion, and remained at a reduced level over 4 months. At the end point, the density of Brn3b-positive RGCs was normal, but their nucleus size was reduced by approximately 24% (P < 001). OCT measurements showed thinning of the inner, but not outer, retina by approximately 9% (P < 0.01) starting 10 to 20 days after lesion. Retinal nerve fiber layer thickness was unchanged. At the end point, retinal homogenates showed a substantial overexpression of BDNF protein level. CONCLUSIONS. Mechanical SC lesion in adult mice results in a rapid, chronic loss of RGC electrical responsiveness that is followed by cell shrinkage but not cell death. The SC-lesion mouse represents a new, inducible model that allows investigating stages and mechanisms of RGC dysfunction without the confounding effects of cell death that are common in the existing models of optic neuropathies and optic nerve lesions. (Invest Ophthalmol Vis Sci. 2013;54:1898‐1904) DOI: 10.1167/iovs.12-11375

We investigated the role of retrograde signaling in the optic nerve on retinal ganglion cell (RGC) electrical responsiveness in the mouse model.Electrical response of RGC was measured by pattern electroretinogram (PERG) in 43 C57BL/6J mice 4 to 6 months old under ketamine/xylazine anesthesia. PERGs were recorded before and at different times after blockade of axon transport with lidocaine at either the retrobulbar level (2 μL, 40 μg/μL) or at level of the superior colliculus (SC, 1 μL, 40 μg/μL). PERGs also were recorded before and at different times after optic nerve crush 1.5 mm behind the eye, followed by TUJ1-positive RGC counts of excised retinas. As controls, PERGs also were recorded after either saline injections or sham optic nerve surgery. The photopic flash electroretinogram (FERG) and visual evoked potential (FVEP) also were recorded before lidocaine and at relevant times afterwards.Lidocaine injection caused rapid (retrobulbar ~10 minutes, SC 1 hour), reversible reduction of PERG amplitude (≥50%). Optic nerve crush caused rapid (10-20 minutes), irreversible reduction of PERG amplitude (70-75%), increase of PERG latency (25%), as well as RGC loss (88%) 1 month after crush. FVEP was unaltered by lidocaine. For all procedures, the FERG was unaltered.As experimental interventions were made at postretinal level(s), PERG changes were likely associated with altered supply of retrogradely-delivered material from the SC. This implies that retrograde transport of target-derived molecules is necessary for normal RGC electrical responsiveness. The time course of early PERG changes is consistent with the speed of fast retrograde axon transport.

To address mitochondrial dysfunction that mediates irreversible visual loss and neurodegeneration of the optic nerve in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis (MS), mice sensitized for EAE were vitreally injected with self-complementary adenoassociated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene that quickly expressed in mitochondria of almost all retinal ganglion cells (RGCs). Visual function assessed by pattern electroretinograms (PERGs) reduced by half in EAE showed no significant reductions with NDI1. Serial optical coherence tomography (OCT) revealed significant inner retinal thinning with EAE that was suppressed by NDI1. Although complex I activity reduced 80% in EAE was not improved by NDI1, in vivo fluorescent probes indicated mitochondrial oxidative stress and apoptosis of the EAE retina were reduced by NDI1. Finally, the 42% loss of axons in the EAE optic nerve was ameliorated by NDI1. Targeting the dysfunctional complex I of EAE responsible for loss of respiration, mitochondrial oxidative stress and apoptosis may be a novel approach to address neuronal and axonal loss responsible for permanent disability that is unaltered by current disease modifying drugs for MS that target inflammation.

To introduce DNA into mitochondria efficiently, we fused adenoassociated virus capsid VP2 with a mitochondrial targeting sequence to carry the mitochondrial gene encoding the human NADH ubiquinone oxidoreductase subunit 4 ( ND4 ). Expression of WT ND4 in cells with the G11778A mutation in ND4 led to restoration of defective ATP synthesis. Furthermore, with injection into the rodent eye, human ND4 DNA levels in mitochondria reached 80% of its mouse homolog. The construct expressed in most inner retinal neurons, and it also suppressed visual loss and optic atrophy induced by a mutant ND4 homolog. The adenoassociated virus cassette accommodates genes of up to ∼5 kb in length, thus providing a platform for introduction of almost any mitochondrial gene and perhaps even allowing insertion of DNA encompassing large deletions of mtDNA, some associated with aging, into the organelle of adults.

Purpose Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed. Methods The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription–polymerase chain reaction (RT–PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy. Results The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT–PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT–PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident. Conclusions Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.

A custom-built OCT system was used to obtain images of the whole mouse eye. We developed a semi-automated segmentation method to detect the boundaries of the anterior and posterior corneal, lens and retinal surfaces as well as the anterior surface of the iris. The radii of curvature of the surfaces were calculated using a conic section fit of each boundary. Image distortions due to refraction of the OCT beam at the successive boundaries were corrected using a ray-tracing algorithm. Corrected ocular distances, radii of curvature of the cornea and lens surfaces, and anterior chamber angle were obtained on 3 C57BL/6J mice. In vivo imaging of the whole eye, segmentation, conic function fits and correction were successful in all three animals. The posterior lens surface of one mouse could not be fit accurately with a conic section. Biometric parameters of C57BL/6J mice compared well with previous published data obtained from histological sections. The study demonstrates the feasibility of quantitative in vivo biometry of mouse models.

Injury to retinal ganglion cell (RGC) axons leads to selective loss of RGCs and vision. Previous studies have shown that exogenous neurotrophic factors promote RGC survival. We investigated the neuroprotective effects of oncostatin M (OSM), a member of the IL-6 family of cytokines, on pattern electroretinogram (PERG) and RGC survival after optic nerve crush (ON-crush) in the mouse. BALB/C mice received ON-crush in the left eyes for either 4-second or 1-second duration (4-s or 1-s). Fluoro-gold retrograde labeling was used to identify RGCs. RGC function was assessed by PERG measurement. OSM or CNTF protein was injected intravitreally immediately after ON-crush. OSM responsive cells were identified by localization of increased STAT3 phosphorylation. Significant higher RGC survival (46% of untreated control) was seen in OSM-treated eyes when assessed 2 weeks after 4-s ON-crush as compared to that (14% of untreated control) of the PBS-treated eyes (P

PURPOSE. To compare the bioelectric field associated with the pattern electroretinogram (PERG) with that of the flash electroretinogram (FERG) in the mouse. METHODS. PERGs and FERGs were recorded from each eye in 32 C57BL/6J mice using corneal silver loops referenced to a subcutaneous needle on the back of the head. PERG stimuli were horizontal gratings of 0.05 cycles per degree and 98% contrast reversing 2 times per second. Light-adapted FERG stimuli were bright strobe flashes. Stimuli were presented either monocularly or binocularly. In some experiments, TTX was injected in one eye and saline in the contralateral eye. RESULTS. The PERG recorded from the contralateral, occluded eye had slightly larger amplitude (1.14 3, P < 0.01) and longer latency (þ1.57 ms, P < 0.01) compared with the ipsilateral eye. Under binocular stimulation, the PERG amplitude was much larger (1.67 3, P < 0.01) than the monocular amplitude. TTX injected in the stimulated eye drastically reduced the PERG in both eyes. Monocular FERGs were recordable from the stimulated eye only and were moderately reduced by TTX. Binocular and monocular FERGs had similar amplitudes. CONCLUSIONS. PERG and FERG generate different bioelectric fields in the mouse. The PERG bioelectric field is consistent with a dipole model whose axis is orthogonal to the eye axis, whereas the standard dipole model for the FERG is coaxial. Possible sources of the PERG bioelectric field are unmyelinated optic nerve axons adjacent to the sclera. Results provide new insights on the generators of the PERG signal and its alterations in mouse models of glaucoma and optic nerve diseases. (Invest Ophthalmol Vis Sci. 2012;53:8086‐8092) DOI:10.1167/ iovs.12-10720

To characterize postnatal changes in eye size in glaucomatous DBA/2J (D2) mice and in nonglaucomatous C57BL/6J mice (B6) in vivo by means of whole-eye optical coherence tomography (OCT).D2 (n = 32) and B6 (n = 36) mice were tested between 2 and 20 months of age in eight age bins. A custom time-domain OCT system with a center wavelength of 825 nm and an axial scan length of 7.1 mm produced axial A-scan interferograms at a rate of 20 A-lines/s with a resolution of 8 μm. Axial length (AL), corneal thickness (CT), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and retinal thickness (RT) were measured in the optical axis and adjusted with corresponding refractive indices. Corneal curvature (CC) and IOP were also measured.AL increased (P0.001) more in the D2 (21%) than in the B6 (9%) mice. There was an interaction effect (two-way ANOVA, P0.001) between age and strain for AL, CT, ACD, and VCD. In the D2 mice, the lens became dislocated posteriorly. Multiple regression analysis in the D2 mice revealed an independent effect of age and IOP (P ≤ 0.01) on axial length. CC steepened in the older D2 mice, whereas it flattened in the B6 mice.In D2 mice, postnatal elongation of AL is larger than that in B6 mice and is associated with a greater increase in ACD and IOP, which seems to be a causal factor. The ease of use, short acquisition time, and noninvasiveness of whole-eye OCT make it suitable for routine use in longitudinal studies of mouse models.

Multiple sclerosis (MS) is a disease associated with the demyelination of neurons.1 MS features an underlying multifactorial etiology2 and presents a spectrum of clinical symptoms.2 Spasm and gait abnormalities are clinical symptoms of demyelinating disease.1,3 MS is frequently associated with visual impairment and vision loss.4 Modification of environmental circumstances, which are often thought to be contributory to the disease process, is severely restricted in human studies compared with studies in animal models. For these reasons, animal models are needed both to understand the underlying pathologic mechanisms and to evaluate novel therapeutic and reparative approaches. It is expected, given the complex nature of the disease and its multifactorial etiology, that a single animal model will not provide insight into all clinical, radiologic, pathologic, and genetic features of MS. In addition to frequently used rodent models,5 nonhuman primates6 and avian models7,8 are used in MS research, which has expanded our understanding of different pathologic aspects of MS. The most commonly studied major categories of animal/rodent models of MS include experimental autoimmune encephalomyelitis (EAE), virally induced (e.g., Theiler's murine encephalomyelitis virus [TMEV] infection) chronic demyelinating models, and toxin-induced models of demyelination such as the cuprizone and focal demyelination induced by lysophosphatidyl choline models.5,9 In addition to the rodent models described, the importance of genetically modified mice cannot be underestimated.10 As stated, vision loss is frequently associated with MS and usually follows a late onset and progressive course.11–13 A number of noninvasive techniques have been used to characterize structural/functional changes in the nervous systems and MS pathology, including functional electrophysiologic measurements,14 optic coherence tomography (OCT),15,16 and magnetic resonance imaging (MRI).11 Electrophysiologic measurements and, in particular, pattern electroretinogram (PERG) help assess functional status and have provided insight about the health of the optic nerve in MS patients.17 A transgenic mouse model (ND4 mice) of MS harboring multiple copies of the DM-20 isoform of myelin proteolipid protein (PLP) is available.18,19 The ND4 mouse is clinically healthy at 3 months of age and has minimal inflammatory signs. Around 3 months of age or immediately thereafter, without the injection of a myelin-specific antigen such as myelin basic protein, ND4 mice spontaneously undergo demyelination. It is, therefore, one of the genetically modified animal models for the study of demyelinating disease and offers intrinsic manifestations of MS symptoms without any external injections. At the same time, littermate controls can be generated to rule out the effects of confounding factors such as aging. MS has been known to be associated with vision loss, but most animal models, including the transgenic ND4 model, remain uncharacterized with respect to the visual system. Here we present a noninvasive characterization of the visual system in this model and extend some of the methods for characterization of the central nervous system. Noninvasive characterization provides the advantage of observing individual animals at different stages of the disease process, not only enabling determination of disease features at the population level but also capturing individual differences and the outcome measures of intervention strategies at individual levels.

Leber hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by acute bilateral loss of central vision.1 In approximately 95% of the cases worldwide, LHON is caused by 3 pathogenic point mutations in mitochondrial DNA (mtDNA) coding for the respiratory chain subunits of complex I genes (the reduced form of nicotinamide adenine dinucleotide–ubiquinone-oxidoreductase), namely m.3460G>A in ND1, m.11778G>A in ND4, and m.14484T>C in ND6.2 Of these 3 mutations, 11778G>A, resulting in an arginine to histidine substitution at amino acid 340, is responsible for half of all LHON cases. Patients with this mutation exhibit the poorest prognosis for spontaneous visual improvement, and there is no effective therapy.3 Most patients with LHON carry the mtDNA mutations in homoplasmic condition, that is, they have no normal ND4 mtDNA. Thus, an effective therapeutic approach would be introduction of normal mtDNA into the affected cells (ganglion cells of the retina) in these patients. One of the major limitations in this aspect is that few practical methods for delivering genes to the mitochondria are available.4,5 To address this, we and other groups adapted an approach termed allotopic expression, in which the mutant mitochondrial gene (in this case ND4) is expressed in the nucleus, translated on cytoplasmic polyribosomes, and transported into mitochondria with the addition of an N-terminal mitochondrial targeting sequence.6-8 Allotopic expression of the normal ND4 gene normalized the defective adenosine triphosphate (ATP) synthesis of LHON cells and improved cell survival.9 Recombinant adeno-associated virus (AAV) vectors have been used safely for ocular gene delivery in a disorder affecting photoreceptors, Leber congenital amaurosis.10,11 Adeno-associated virus is derived from the nonpathogenic virus belonging to the Dependovirus genus of the Parvoviridae family and contains a linear single-stranded DNA genome enclosed in a capsid composed of 3 proteins: VP1, VP2, and VP3.12,13 Studies on the hybrid vectors or the pseudotyped vectors containing the promoter and the trans-gene flanked by the 145–base pair (bp) AAV inverted terminal repeats packaged into different capsid serotypes not only reveal cellular tropism in different organs but also show an increased efficiency for specific tissues.14,15 In a previous study, we showed that the human ND4 subunit was expressed in only one-third of retinal ganglion cells (RGCs) 1 month after intravitreal injections in mice. For treatment of acute visual loss in LHON, a more efficient and rapid gene expression delivery system is needed. Strategies to improve the efficiency of transgene expression are being continuously explored by manipulating the AAV genome. One such development is the self-complementary AAV (scAAV) vector with a double-stranded vector genome. This vector was generated by deleting the terminal resolution site from one of the inverted terminal repeats of recombinant AAV (rAAV), which prevents the initiation of replication at the mutated end.16 This strategy allows packaging of a self-complementary form of vector DNA that has the ability of annealing to itself to form a double-stranded DNA immediately on vector delivery to the target cell nucleus. This overcomes the rate-limiting step of replication of the single-stranded viral genome into the double-stranded DNA within the cell nucleus, thereby increasing the efficiency and speed of transgene expression manyfold, as observed in the liver,17,18 brain,19 eye,20,21 and heart.22 Whether scDNA is single or double stranded while inside the AAV vector capsid is unclear at present. In this study, we compared expression of the human ND4 subunit gene when delivered by scAAV relative to that when delivered by the single-stranded AAV (ssAAV).

AG-to-A transition at nucleotide 11778 in mitochondrial DNA (mtDNA) in the gene specifying the NADH dehydrogenase subunit 4 (ND4) of complex I, which results in an arginine-to-histidine substitution at amino acid 340, is responsible for half of the cases of Leber hereditary optic neuropathy (LHON), a disease that causes blindness in young adults.1-3 Moreover, patients with the G11778A mutation in mtDNA have the poorest prognosis for spontaneous visual improvement. There is no effective therapy for LHON or, for that matter, for any other disease caused by mutated mtDNA. Unlike the other mitochondrial diseases in which the ratio of mutated to normal mtDNA determines phenotype (a condition called heteroplasmy), visual loss from LHON usually requires 100% mutated mtDNA (homoplasmy). Therefore, it is highly likely that the introduction of normal mtDNA into patients with LHON may protect against visual loss and blindness. Any proposed therapy for LHON or other diseases caused by mutated mtDNA must wrestle with several problems: There is no way yet to correct mutated mtDNA in living animals; there is no delivery system for DNA into mitochondria; there is no confirmed evidence for recombination between mtDNA molecules that would enable gene replacement; and there are no selectable markers for mitochondrial transformation. We and others4,5 have taken an alternative approach for correcting point mutations in mitochondrial protein-coding genes by expressing these genes in the nucleus in conjunction with an N-terminal targeting sequence for mitochondrial import. Most mitochondrial proteins are, in fact, made this way and are transported into the organelles after their translation on cytoplasmic polyribosomes. There are at least two major obstacles to correcting mitochondrial mutations in this way. First, mitochondrial translation makes use of a genetic code partially different from that of the nuclear-cytoplasmic system. Second, some mitochondrial proteins may be too hydrophobic to be imported and properly assembled from the “outside.” The first obstacle can be addressed by completely recoding mitochondrial genes so that they can be expressed on cytoplasmic ribosomes,6,7 an approach coined allotopic expression. This process involves using overlapping oligonucleotides to rebuild the genes in the universal genetic code, choosing codons that permit high-level translation,8 or using in situ mutagenesis to change codons that differ between the mitochondria and the nucleus.9 The second problem is more difficult. Claros et al.10 attempted import of a universal code version of apocytochrome b from the nucleus of yeast and found that each of the eight transmembrane helices of this protein could be transported separately into mitochondria but that no more than three or four of the hydrophobic domains could be transported as one peptide. They10 and others11,12 argue that the hydrophobicity of mitochondrial proteins imposes a limit on which genes could be transplaced to the nucleus, that allotopically expressed proteins do not integrate into host respiratory complexes, and that allotopically expressed proteins result in significant cytotoxicity, even inducing cell death. Rather than continue this debate in cultured cells, here we evaluated the potential value or pitfalls of allotopic expression of a normal human ND4 gene in the mouse visual system, in which we previously demonstrated that allotopic expression of the mutant human R340H ND4 induced optic disc edema with the demise of retinal ganglion cells (RGCs) and axons of the optic nerve.13

SummaryNeisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.