Tetrameric UvrD Helicase Is Located at the E. Coli Replisome due to Frequent Replication Blocks.

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

Test-Retest Reliability of Virtual Acoustic Space Identification Test in School-Going Children.

PURPOSE: The virtual acoustic space identification (VASI) test was designed to assess spatial-hearing acuity by simulating sound location perception in a closed field (under headphones). The utility of this tool in children can be asserted only if the test results are consistent across measurement sessions, which is evaluated in this study using test-retest reliability assessments. METHOD: The VASI test assessed the spatial abilities of 40 typically developing school-aged children aged 7-13 years (M age = 10.47 ± 1.83 years, 22 boys, 18 girls). The test consisted of eight virtual location percepts (with 45° separation) produced under headphones (Sennheiser HD 569). Each spatial percept was presented randomly 7 times at 65 dB SPL. Each participant completed the assessment in three measurement sessions (baseline, intrasession, and intersession). The accuracy scores at each location and overall accuracy scores were compared across the sessions. RESULTS: The Shapiro-Wilk test indicated that the VASI data were not normally distributed. Intraclass correlation coefficient analysis revealed excellent test-retest reliability of the overall accuracy scores and moderate-to-high reliability of location-specific scores. This was complimented by the low response variability of the overall and location-specific accuracy scores. The Bland-Altman analysis also indicated minimal bias in VASI accuracy scores across the three sessions. CONCLUSIONS: It can be concluded from the results that VASI is a reliable tool for assessing spatial-hearing acuity in school-aged children. The high test-retest reliability and ease of portability make the test highly relevant for classroom setups where early diagnosis and intervention of spatial deficits can play a critical role in determining the academic success of school-going children.

Age differences in binaural and working memory abilities in school-going children.

OBJECTIVES: Binaural hearing is the interplay of acoustic cues (interaural time differences: ITD, interaural level differences: ILD, and spectral cues) and cognitive abilities (e.g., working memory, attention). The current study investigated the effect of developmental age on auditory binaural resolution and working memory and the association between them (if any) in school-going children. METHODS: Fifty-seven normal-hearing school-going children aged 6-15 y were recruited for the study. The participants were divided into three groups: Group 1 (n=17, Mage = 7.1y ± 0.72 y), Group 2 (n = 23; Mage = 10.2y ± 0.8 y), Group 3 (n = 17; Mage: 14.1 y ±1.3 y). Group 4, with normal hearing young adults (n = 20; Mage = 21.1 y± 3.2 y), was included for comparing the maturational changes in former groups with adult values. Tests of binaural resolution (ITD and ILD thresholds) and auditory working memory (forward and backward digit span and 2n-back digit) were administered to all the participants. RESULTS: Results indicated a main effect of age on spatial resolution and working memory, with the median of lower age groups (Group 1 & Group 2) being significantly poorer (p < 0.01) than the higher age groups (Group 3 & Group 4). Groups 2, 3, and 4 performed significantly better than Group 1 (p < 0.001) on the forward span and ILD task. Groups 3 and 4 had significantly better ITD (p = 0.04), backward span (p = 0.02), and 2n-back scores than Group 2. A significant correlation between scores on working memory tasks and spatial resolution thresholds was also found. On discriminant function analysis, backward span and ITD emerged as sensitive measures for segregating older groups (Group 3 & Group 4) from younger groups (Group 1 & Group 2). CONCLUSIONS: The present study showed that the ILD thresholds and forward digit span mature by nine years. However, the backward digit span score continued to mature beyond 15 y. This finding can be attributed to the influence of auditory attention (a working memory process) on the binaural resolution, which is reported to mature till late adolescence.

Comparison of a Free-Field and a Closed-Field Sound Source Identification Paradigms in Assessing Spatial Acuity in Adults With Normal Hearing Sensitivity

Background and Objectives@#Traditional sound field localization setups in a free-field environment closely represent real-world situations. However, they are costly and sophisticated, and it is difficult to replicate similar setups in every clinic. Hence, a cost-effective, portable, and less sophisticated virtual setup will be more feasible for assessing spatial acuity in the clinical setting. The virtual auditory space identification (VASI) test was developed to assess spatial acuity using virtual sources in a closed field. The present study compares the legitimacy of these two methods. @*Subjects and Methods@#Fifty-five individuals with normal hearing (mean age±SD: 21± 3.26 years) underwent spatial acuity assessment using two paradigms: 1) the sound field paradigm (localization test) and 2) the virtual paradigm (VASI test). Location-specific and overall accuracy scores and error rates were calculated using confusion matrices for each participant in both paradigms. @*Results@#The results of Wilcoxon signed-rank tests showed that the locationspecific and overall accuracy scores for both paradigms were not significantly different. Further, both paradigms did not yield significantly different localization error rates like right and left intra-hemifield errors, inter-hemifield errors, and front-back errors. Spearman’s correlation analysis showed that all the measures of the two paradigms had mild to moderate correlation. @*Conclusions@#These results demonstrate that both VASI and the sound field paradigm localization test performed equally well in assessing spatial acuity.

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity.

The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA. RecBCD is a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks. It also facilitates coating of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA or RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA cross-linker mitomycin C, we induce DNA damage and quantify the resulting steady state changes in stoichiometry, cellular protein copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA stoichiometries equivalent to several hundred molecules that assemble largely in dimeric subunits before DNA damage, but form periodic subunits of approximately 3-4 molecules within mature filaments of several thousand molecules. Unexpectedly, we find that the physiologically predominant forms of RecB are not only rapidly diffusing monomers, but slowly diffusing dimers.

Long-term Visual and Anatomical Outcome after Surgical Management of Epiretinal Membrane.

OBJECTIVE: To assess the functional and surgical long-term outcomes following epiretinal membrane surgery. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Layton Rahmatulla Benevolent Trust Tertiary Care Eye Hospital, Karachi, from January 2016 to December 2020. METHODOLOGY: A medical record review was carried out of patients, who had undergone surgical management of ERM and had presented for follow-up for at least three years. Best corrected visual acuity (BCVA) and Optical Coherence Tomography (OCT) parameter [(integrity of ellipsoid zone (EZ)] was evaluated. BCVA, using Snellen's chart, is performed routinely on each visit, which was converted to logMAR chart for analysis in this study. OCT was also performed to evaluate the integrity of outer retinal layers using Heidelberg OCT. RESULTS: Sixty-five eyes of 54 patients were included in the study, including 41 eyes of 36 men (63%) and 24 eyes of 18 women (37%). Mean age was 46.2 ± 8.9 years. The mean BCVA significantly improved from 0.88 ± 0.28 logMAR (6/45 Snellen) preoperatively to 0.64 ± 0.21 (6/27) at the end of first year (p <0.001), which improved to 0.54 ± 0.19 (6/21) at the end of second year, and 0.53 ± 0.20 (6/20) after three years of follow-up. The post-op vision at three years was stratified according to the integrity of EZ on OCT performed at the same follow-up; and a significant difference was observed. EZ was intact in 52 eyes with a mean BCVA of 0.49 ± 0.16 logMAR (6/18 Snellen); while it was found disrupted in 13 eyes, where the BCVA was 0.68 ± 0.26 (6/29). CONCLUSION: Anatomically intact outer retinal layer significantly correlated with improved BCVA. Key Words: Epiretinal membrane, Optical coherence tomograghy, Best corrected visual acuity.

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics.

Each cell division requires the complete and accurate duplication of the entire genome. In bacteria, the duplication process of the often-circular chromosomes is initiated at a single origin per chromosome, resulting in two replication forks that traverse the chromosome in opposite directions. DNA synthesis is completed once the two forks fuse in a region diametrically opposite the origin. In some bacteria, such as Escherichia coli, the region where forks fuse forms a specialized termination area. Polar replication fork pause sites flanking this area can pause the progression of replication forks, thereby allowing forks to enter but not to leave. Transcription of all required genes has to take place simultaneously with genome duplication. As both of these genome trafficking processes share the same template, conflicts are unavoidable. In this review, we focus on recent attempts to add additional origins into various ectopic chromosomal locations of the E. coli chromosome. As ectopic origins disturb the native replichore arrangements, the problems resulting from such perturbations can give important insights into how genome trafficking processes are coordinated and the problems that arise if this coordination is disturbed. The data from these studies highlight that head-on replication-transcription conflicts are indeed highly problematic and multiple repair pathways are required to restart replication forks arrested at obstacles. In addition, the existing data also demonstrate that the replication fork trap in E. coli imposes significant constraints to genome duplication if ectopic origins are active. We describe the current models of how replication fork fusion events can cause serious problems for genome duplication, as well as models of how such problems might be alleviated both by a number of repair pathways as well as the replication fork trap system. Considering the problems associated both with head-on replication-transcription conflicts as well as head-on replication fork fusion events might provide clues of how these genome trafficking issues have contributed to shape the distinct architecture of bacterial chromosomes.

Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome.

DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live Escherichia coli cells. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.

Inhibiting translation elongation can aid genome duplication in Escherichia coli.

Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication.

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair.

Single-Molecule Observation of DNA Replication Repair Pathways in E. coli.

The method of action of many antibiotics is to interfere with DNA replication-quinolones trap DNA gyrase and topoisomerase proteins onto DNA while metronidazole causes single- and double-stranded breaks in DNA. To understand how bacteria respond to these drugs, it is important to understand the repair processes utilised when DNA replication is blocked. We have used tandem lac operators inserted into the chromosome bound by fluorescently labelled lac repressors as a model protein block to replication in E. coli. We have used dual-colour, alternating-laser, single-molecule narrowfield microscopy to quantify the amount of operator at the block and simultaneously image fluorescently labelled DNA polymerase. We anticipate use of this system as a quantitative platform to study replication stalling and repair proteins.

Recombination and replication.

The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.

23-gauge sutureless vitreo-retinal surgery for superior rhegmatogenous retinal detachment.

OBJECTIVE: To determine the results of 23-gauge sutureless vitreo-retinal surgery for superior/supero-temporal rhegmatogenous retinal detachment (RRD). STUDY DESIGN: Quasi experimental study. PLACE AND DURATION OF STUDY: LRBT, Free Base Eye Hospital, Karachi, from January 2010 to December 2011. METHODOLOGY: Adult patients who underwent 23-gauge sutureless vitreo-retinal surgery along with use of Perfluoropropane (C3F8) gas as internal tamponading agent for fresh (upto 3 weeks) superior/supero-temporal RRD was reviewed. Major outcome measures were anatomical success, best corrected visual acuity (BCVA) with Log Mar and complications during and after surgery. Postoperative follow-up was done on 1st day and at 1st, 4th, 8th and finally at 12th week. RESULTS: Sixty eyes of 60 patients, age between 30 - 60 years including 37 (61.67%) males and 23 (38.33%) females having superior or superatemporal RRD underwent 23-guage sutureless vitreo-retinal surgery with the use of perfluoropropane (C3 F8) gas as internal temponade at the end of procedure. Anatomical success rate was 81.66% (49 out of 60 eyes) with first surgery and raised to 90% (54 cases) with second surgery. Log Mar BCVA significantly improved from mean baseline 0.93 to 0.49 with mean difference of 0.43 (p < 0.001), 95% confidence interval. Postoperative complications were sub-conjunctival haemorrhage in 11 eyes (18.33%), wound leak in 7 eyes (11.66%), anterior chamber became shallow in 6 eyes (10%), cataract developed in 5 eyes (8.33%), re-retinal detachment in 4 eyes (6.66%), ocular hypotony and sterile inflammatory reaction in 3 eyes (5%) each, while iatrogenic breaks developed in 2 eyes (3.33%). CONCLUSION: The 23-gauge sutureless vitreo-retinal surgery for superior rhegmatogenous retinal detachment achieved high anatomical success and significant visual improvement. Sub-conjunctival haemorrhage was the most frequent procedural complication.

Management of Macular Pre-Retinal Subhyaloid hemorrhage by Nd:Yag laser hyaloidotomy.

OBJECTIVE: To evaluate the efficacy, visual outcome and complication following Nd:Yag laser hyaloidtomy for subhyaloid hemorrhage. METHODS: This interventional case series was managed at LRBT, Free Base Eye Hospital Karachi from January 2010 to December 2010. It included 30 eyes of 30 patients with subhyaloid hemorrhage due to different causes which underwent Nd: Yag laser sublyaloidotomy Results: Out of thirty patients, eighteen (60%) were male and twelve (40%) were females. Mean age was 32.57 years. Males pre-dominated the study. Pre laser visual acuity was between counting finger at one meter in 22 patients (73.33%) and between counting finger one meter to hand movement in 8 patients (26.66%). Vision improved to 6/6 in 10 patients (33%), 6/9 - 6/12 in 17 patients (56.66%) and between 6/24 - 6/60 in 3 patients (9.99%) at the end of follow up. Complications were persistent vitreous hemorrhage in one (3.33%) patient, failed drainage in one (3.33%) patient and metamorphopsia in one (3.33%) patient. CONCLUSION: Nd: Yag laser hyloidotomy is an excellent technique for management of Subhyaloid hemorrhage with early visual recovery provided there is no macular pathology.

Human ophthalmomyiasis externa caused by the sheep botfly Oestrus ovis: A case report from Karachi, Pakistan

Ocular myiasis due to Oestrus ovis larvae infestation is an eye infection in humans. A case of ophthalmomyiasis externa in a young male from Karachi, Pakistan in winter (December 2012), without history of close proximity to domestic animals or visit to any rural area was reported. The condition is self-limiting and the disease is confined to the conjunctiva. The eye was locally anesthetized and washed with 5% povidine iodine solution. A total number of 27 first instar larvae of Oestrus ovis were removed with fine forceps. The patient received 0.5% moxifloxacin and diclofenac eye drops for one week. His eye was examined after one day, one week and one month and the recovery status was favorable. The present case raise the awareness among ophthalmologists regarding larval conjunctivitis as one of the causes of conjunctivitis and it can occur throughout the year in any season including winter. Moreover, it can occurr in any area either rural or urban with or without close proximity to domestic animals especially in subtropical regions with high parasitic burden.

Outcomes of silicone oil removal.

OBJECTIVE: To evaluate the outcome and complications of removal of silicone oil after pars plana vitrectomy. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Layton Rahmatullah Benevolent Trust (L.R.B.T), Free Base Eye Hospital, Karachi, from February 2008 to January 2011. METHODOLOGY: Ninety five eyes of 95 patients with a history of undergoing three-port pars plana vitrectomy were included in this study that subsequently underwent removal of silicone oil. Silicone oil was removed after ophthalmoscopically determining retina attachment or when the duration of silicone oil tamponade was atleast of 6 months. Patients were followed for a period of 12 months. RESULTS: Retinal re-detachment was seen in 19 (20%) out of 95 eyes, vitreous haemorrhage in 2 (2.1%) out of 95 eyes, corneal decompensation in 6 (6.3%) out of 95 eyes, hypotony in 7 (7.3%) out of 95 eyes, phthisis bulbi in 2 (2.1%) out of 95 eyes and lens opacification in 9 (9.4%) out of 95 eyes. CONCLUSION: In this study, silicone oil removal resulted in various complications among which retinal re-detachment was the most frequent.

Rho-dependent transcription termination is essential to prevent excessive genome-wide R-loops in Escherichia coli.

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translation-uncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.

Visual outcome of pars plana vitrectomy for dropped nucleus after phacoemulsification.

OBJECTIVE: To determine the visual outcome of patients who underwent pars plana vitrectomy for dropped nucleus after phacoemulsification. STUDY DESIGN: Interventional case series. PLACE AND DURATION OF STUDY: LRBT Free Base Eye Hospital, Karachi, from February 2008 to January 2011. METHODOLOGY: Forty-eight eyes of forty eight patients having history of dropped nucleus (soft remnant, half nucleus or complete nucleus) underwent 20 gauge pars plana vitrectomy within 24 days of phacoemulsification. After complete vitrectomy nucleus was lifted with the help of perfluorocarbon and removed either through a limbal incision or by using phacofragmenter, whereas small lens remnants were removed with a vitreous cutter. Intraocular lens was implanted at the end of surgery. Postoperative visual acuity, and any complications were assessed. Patients were followed for a period of 12 months. RESULTS: Final visual acuity ranged from 6/9 to 6/18 in 34 eyes (70.83%), 6/24 to 6/36 in 8 eyes (16.66%) and 6/60 or less in 6 of 48 eyes (12.5%). Complications included raised intraocular pressure in 6 eyes (12.5%) and retinal detachment in 2 eyes (4.1%), corneal oedema and decompensation in 3 eyes (6.25%) and cystoids macular oedema in 4 cases (8.33%) out of 48 cases. CONCLUSION: The loss of crystalline lens in the vitreous during phacoemulsification is a severe complication, but appropriate and timely management can restore good visual outcome and minimize complications.