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1.  In vivo snapshot hyperspectral image analysis of age-related macular degeneration 
Drusen, the hallmark lesions of age related macular degeneration (AMD), are biochemically heterogeneous and the identification of their biochemical distribution is key to the understanding of AMD. Yet the challenges are to develop imaging technology and analytics, which respect the physical generation of the hyperspectral signal in the presence of noise, artifacts, and multiple mixed sources while maximally exploiting the full data dimensionality to uncover clinically relevant spectral signatures. This paper reports on the statistical analysis of hyperspectral signatures of drusen and anatomical regions of interest using snapshot hyperspectral imaging and non-negative matrix factorization (NMF). We propose physical meaningful priors as initialization schemes to NMF for finding low-rank decompositions that capture the underlying physiology of drusen and the macular pigment. Preliminary results show that snapshot hyperspectral imaging in combination with NMF is able to detect biochemically meaningful components of drusen and the macular pigment. To our knowledge, this is the first reported demonstration in vivo of the separate absorbance peaks for lutein and zeaxanthin in macular pigment.
PMCID: PMC4167017  PMID: 21096261
2.  Potential roles for DNA replication and repair functions in cell killing by streptomycin 
Mutation research  2013;749(0):87-91.
The aminoglycoside streptomycin binds to ribosomes to promote mistranslation and eventual inhibition of translation. Streptomycin kills bacteria, whereas many other non-aminoglycoside inhibitors of translation do not. Because mistranslation is now known to affect DNA replication, we asked if hydroxyurea, a specific inhibitor of DNA synthesis, affects killing, and find that hydroxyurea significantly attenuates killing by streptomycin. We find that the hydroxyl radical scavengers D-mannitol and thiourea have either no effect or only a modest protective effect. The iron chelator 2,2′-dipyridyl eliminated killing by streptomycin, but further investigation revealed that it blocks streptomycin uptake. Prior treatment of cells with low-levels of methyl methanesulfonate to induce the adaptive response to alkylation leads to a significant attenuation of killing, which, together with the hydroxyurea effect, suggests roles for DNA replication and repair functions in cell killing by streptomycin.
PMCID: PMC3788604  PMID: 23958411
mistranslation; DNA replication and repair; dipyridyl; antibiotic uptake; hydroxyl radicals; DNA alkylation damage; alkB
3.  SOS induction and mutagenesis by dnaQ missense alleles in wild type cells 
Mutation Research  2012;735(1-2):46-50.
Mistranslation leads to elevated mutagenesis and replication arrest, both of which are hypothesized to result from the presence of mixed populations of wild type and mistranslated versions of DNA polymerase III subunit proteins. Consistent with this possibility, expression of missense alleles of dnaQ (which codes for the proofreading subunit ε in wild type (dnaQ+) cells is shown to lead to SOS induction as well as mutagenesis. Exposure to sublethal concentrations of streptomycin, an aminoglycoside antibiotic known to promote mistranslation, also leads to SOS induction.
PMCID: PMC3389301  PMID: 22677460
DNA replication; SOS induction; Mutagenesis; Mistranslation; Streptomycin; Mutator
4.  Factors Affecting Perceptual Threshold in Argus II Retinal Prosthesis Subjects 
The Argus II epiretinal prosthesis has been developed to provide partial restoration of vision to subjects blinded from outer retinal degenerative disease. Participants were surgically implanted with the system in the United States and Europe in a single arm, prospective, multicenter clinical trial. The purpose of this investigation was to determine which factors affect electrical thresholds in order to inform surgical placement of the device.
Electrode–retina and electrode–fovea distances were determined using SD-OCT and fundus photography, respectively. Perceptual threshold to electrical stimulation of electrodes was measured using custom developed software, in which current amplitude was varied until the threshold was found. Full field stimulus light threshold was measured using the Espion D-FST test. Relationships between electrical threshold and these three explanatory variables (electrode–retina distance, electrode–fovea distance, and monocular light threshold) were quantified using regression.
Regression analysis showed a significant correlation between electrical threshold and electrode–retina distance (R2 = 0.50, P = 0.0002; n = 703 electrodes). 90.3% of electrodes in contact with the macula (n = 207) elicited percepts at charge densities less than 1 mC/cm2/phase. These threshold data also correlated well with ganglion cell density profile (P = 0.03). A weaker, but still significant, inverse correlation was found between light threshold and electrical threshold (R2 < 0.52, P = 0.01). Multivariate modeling indicated that electrode–retina distance and light threshold are highly predictive of electrode threshold (R2 = 0.87; P < 0.0005).
Taken together, these results suggest that while light threshold should be used to inform patient selection, macular contact of the array is paramount.
Translational Relevance
Reported Argus II clinical study results are in good agreement with prior in vitro and in vivo studies, and support the development of higher-density systems that employ smaller diameter electrodes. ( identifier: NCT00407602)
PMCID: PMC3763895  PMID: 24049718
retinal prosthesis; retinal degeneration; retinitis pigmentosa
5.  Blind subjects implanted with the Argus II retinal prosthesis are able to improve performance in a spatial-motor task 
To determine to what extent subjects implanted with the Argus II retinal prosthesis can improve performance compared with residual native vision in a spatial-motor task.
High-contrast square stimuli (5.85 cm sides) were displayed in random locations on a 19″ (48.3 cm) touch screen monitor located 12″ (30.5 cm) in front of the subject. Subjects were instructed to locate and touch the square centre with the system on and then off (40 trials each). The coordinates of the square centre and location touched were recorded.
Ninety-six percent (26/27) of subjects showed a significant improvement in accuracy and 93% (25/27) show a significant improvement in repeatability with the system on compared with off (p<0.05, Student t test). A group of five subjects that had both accuracy and repeatability values <250 pixels (7.4 cm) with the system off (ie, using only their residual vision) was significantly more accurate and repeatable than the remainder of the cohort (p<0.01). Of this group, four subjects showed a significant improvement in both accuracy and repeatability with the system on.
In a study on the largest cohort of visual prosthesis recipients to date, we found that artificial vision augments information from existing vision in a spatial-motor task.
Clinical trials registry no
PMCID: PMC3345188  PMID: 20881025
6.  Improvement after transvitreal limited arteriovenous crossing manipulation without vitrectomy for complicated branch retinal vein occlusion using 25 gauge instrumentation 
PMCID: PMC1772717  PMID: 15965182
retinal pick; arteriovenous adventitial sheathotomy; branch retinal vein occlusion; transvitreal arteriovenous- crossing manipulation without vitrectomy; optical coherence tomography
7.  Escherichia coli Cells Bearing a Ribosomal Ambiguity Mutation in rpsD Have a Mutator Phenotype That Correlates with Increased Mistranslation 
Journal of Bacteriology  2003;185(16):5015-5018.
Escherichia coli cells bearing certain mutations in rpsD (coding for the 30S ribosomal protein S4) show a ribosomal ambiguity (Ram) phenotype characterized by increased translational error rates. Here we show that spontaneous mutagenesis increases in Ram cells bearing the rpsD14 allele, suggesting that the recently described translational stress-induced mutagenesis pathway is activated in Ram cells.
PMCID: PMC166475  PMID: 12897024
8.  Intraocular retinal prosthesis. 
PURPOSE: An electronic implant that can bypass the damaged photoreceptors and electrically stimulate the remaining retinal neurons to restore useful vision has been proposed. A number of key questions remain to make this approach feasible. The goal of this thesis is to address the following 2 specific null hypotheses: (1) Stimulus parameters make no difference in the electrically elicited retinal responses. (2) Just as we have millions of photoreceptors, so it will take a device that can generate millions of pixels/light points to create useful vision. METHODS: For electrophysiologic experiments, 2 different setups were used. In the first setup, charge-balanced pulses were delivered to the retinal surface via electrodes inserted through an open sky approach in normal or blind retinal degenerate (rd) mice. In the second setup, the rabbit retina was removed under red light conditions from an enucleated eye and then maintained in a chamber while being superfused with oxygenated, heated Ames media. In both setups, stimulating electrodes and recording electrodes were positioned on the retinal surface to evaluate the effect of varying stimulation parameters on the orthodromic retinal responses (i.e., recording electrode placed between stimulating electrodes and optic nerve head). For psychophysical experiments, visual images were divided into pixels of light that could be projected in a pattern on the retina in up to 8 sighted volunteers. Subjects were asked to perform various tasks ranging from reading and face recognition to various activities of daily living. RESULTS: Electrophysiologic experiments: In a normal mouse, a single cycle of a 1-kHz sine wave was significantly more efficient than a 1-kHz square wave (P < .05), but no such difference was noted in either of the 8- or 16-week-old rd mouse groups (8-week-old, P = .426; 16-week-old, P = .078). Charge threshold was significantly higher in 16-week-old rd mouse versus both 8-week-old rd and normal mouse for every stimulus duration (P < .05). In all groups, short duration pulses (40, 80, and 120 microseconds) were more efficient in terms of total charge (the product of pulse amplitude and pulse duration) than longer (500 and 1,000 microseconds) pulses (P < .05). In all groups, applying a pulse train did not lead to more efficient charge usage (P < .05). Psychophysical experiments: In high-contrast tests, facial recognition rates of over 75% were achieved for all subjects with dot sizes of up to 31.5 minutes of arc when using a 25 x 25 grid with 4.5 arc minute gaps, a 30% dropout rate, and 6 gray levels. Even with a 4 x 4 array of pixels, some subjects were able to accurately describe 2 of the objects. Subjects who were able to read the 4-pixel letter height sentences (on the 6 x 10 and 16 x 16 array) seemed to have a good scanning technique. Scanning at the proper velocity tends to bring out more contrast in the lettering. The reading speed for the 72-point font is a bit slower than for the next smaller font. This may be due to the limited number of letters (3) visible in the window with this large font. CONCLUSIONS: Specific parameters needed to stimulate the retina were identified. Delineating the optimum parameters will decrease the current requirements. Psychophysical tests show that with limited pixels and image processing, useful vision is possible. Both these findings should greatly simplify the engineering of an electronic retinal prosthesis.
PMCID: PMC1359018  PMID: 11797315
9.  Requirement for Homologous Recombination Functions for Expression of the mutA Mistranslator tRNA-Induced Mutator Phenotype in Escherichia coli 
Journal of Bacteriology  2000;182(5):1427-1431.
Expression of the Escherichia coli mutA mutator phenotype requires recA, recB, recC, ruvA, and ruvC gene, but not recD, recF, recO, or recR genes. Thus, the recBCD-dependent homologous recombination system is a component of the signal pathway that activates an error-prone DNA polymerase in mutA cells.
PMCID: PMC94434  PMID: 10671469
10.  Multiplex Sequence Analysis Demonstrates the Competitive Growth Advantage of the A-to-G Mutants of Clarithromycin-Resistant Helicobacter pylori 
Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.
PMCID: PMC89182  PMID: 10049289
11.  SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions 
Journal of Bacteriology  1999;181(5):1515-1523.
Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.
PMCID: PMC93541  PMID: 10049383
12.  Escherichia coli Cells Exposed to Streptomycin Display a Mutator Phenotype 
Journal of Bacteriology  1999;181(3):1043-1044.
Mistranslation mediated by the mutA and mutC tRNA alleles elicits a strong mutator phenotype (H. S. Murphy and M. Z. Humayun, J. Bacteriol. 179:7507–7514, 1997; M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380–4385, 1996). Here, we show that exposure to streptomycin, an antibiotic known to promote mistranslation, induces a recA- and umuDC-independent mutator phenotype detected as enhanced mutagenesis at a 3,N4-ethenocytosine lesion borne on transfected M13 single-stranded DNA.
PMCID: PMC93477  PMID: 9922274
14.  Escherichia coli cells expressing a mutant glyV (glycine tRNA) gene have a UVM-constitutive phenotype: implications for mechanisms underlying the mutA or mutC mutator effect. 
Journal of Bacteriology  1997;179(23):7507-7514.
Transfection of M13 single-stranded viral DNA bearing a 3,N4-ethenocytosine lesion into Escherichia coli cells pretreated with UV results in a significant elevation of mutagenesis at the lesion site compared to that observed in untreated cells. This response, termed UVM, for UV modulation of mutagenesis, is induced by a variety of DNA-damaging agents and is distinct from known cellular responses to DNA damage, including the SOS response. This report describes our observation, as a part of our investigation of the UVM phenomenon, that E. coli cells bearing a mutA or mutC allele display a UVM-constitutive phenotype. These mutator alleles were recently mapped (M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380-4385, 1996) to the glyV (mutA) and glyW (mutC) tRNA genes. Each mutant allele was shown to arise by an identical mutation in the anticodon sequence such that the mutant tRNAs could, in principle, mistranslate aspartate codons in mRNA as glycine at a low level. Because a UVM-constitutive phenotype resulting from a mutation in a tRNA gene was unexpected, we undertook a series of experiments designed to test whether the phenotype was indeed mediated by the expression of mutant glycine tRNAs. We placed either a wild-type or a mutant glyV gene under the control of a heterologous inducible promoter on a plasmid vector. E. coli cells expressing the mutant glyV gene displayed all three of the following phenotypes: (i) missense suppression of a test allele, (ii) a mutator phenotype measured by mutation to rifampin resistance, and (iii) a UVM-constitutive phenotype. These phenotypes were not associated with cells expressing the wild-type glyV gene or with cells in which the mutant allele was present but was not transcriptionally induced. These observations provide strong support for the idea that expression of mutant tRNA can confer a mutator phenotype, including the UVM-constitutive phenotype observed in mutA and mutC cells. However, our data imply that low-level mistranslation of the epsilon subunit of polymerase III probably does not account for the observed UVM-constitutive phenotype. Our results also indicate that mutA deltarecA double mutants display a normal UVM phenotype, suggesting that the mutA effect is recA dependent. The observations reported here raise a number of intriguing questions and raise the possibility that the UVM response is mediated through transient alteration of the replication environment.
PMCID: PMC179703  PMID: 9393717
15.  Role of mismatch repair in the Escherichia coli UVM response. 
Journal of Bacteriology  1996;178(23):6651-6657.
Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.
PMCID: PMC178557  PMID: 8955278
16.  Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response. 
Journal of Bacteriology  1995;177(21):6041-6048.
The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.
PMCID: PMC177440  PMID: 7592365
17.  Antibodies specific to a deoxyribodinucleotide sequence. 
Nucleic Acids Research  1977;4(9):2997-3006.
Antibodies were raised in rabbits against the bovine serum albumin conjugate of dpApT. Analysis by double diffusion in agar gel and quantitative precipitation test showed the presence of antibodies specific to the hapten in the antisera. Quantitative data on the specificity of the antibodies were obtained by studying the inhibition of the binding of 3H-dpApT to the antisera by various nonradioactive mono- and oligonucleotides, using a nitrocellulose membrane binding assay. The antibodies were found to be highly specific for the dinucleotide sequence dpApT. The antibodies were able to bind to synthetic oligonucleotides containing the sequence dpApT and to denatured calf thymus DNA.
PMCID: PMC342629  PMID: 410004

Results 1-17 (17)