Search

Zhu Li Yap,1,2 Sushma Verma,2 Yi Fang Lee,1,2 Charles Ong,1,2 Aditi Mohla,1,2 Shamira A Perera1,2 1Singapore National Eye Center, 2Singapore Eye Research Institute, Singapore Abstract: There are two long-standing theories about the pathogenesis of glaucoma – barotrauma and the effect of vascular hypoxia. Currently, it is still unknown whether diminished blood flow is the cause or result of glaucomatous atrophy of ganglion cells and the optic nerve. Though many other imaging techniques used to directly assess ocular blood flow have been well studied, they are limited by their inability to directly assess metabolism in the ocular tissues or measure the oxygen carrying capacity in the vessels. Retinal oximetry is a relatively novel, noninvasive imaging technique that reliably measures oxygen saturation levels in the retinal vessels, offering surrogate markers for the metabolic demands of the eye. The clinical significance of these measurements has not been well established. Thus, this review gives an overview of ocular imaging and current retinal oximetry techniques, while contextualizing the important oximetry studies that have investigated the vascular theory behind glaucoma. Keywords: retinal oximetry, glaucoma, Oxymap, retinal vasculature, glaucoma imaging

Zhu Li Yap,1,2 You Chuen Chin,1 Judy Yu-Fen Ku,1 Tat Keong Chan,1,2 Gillian Teh,1,2 Monisha Esther Nongpiur,2,3 Tin Aung,1,2 Shamira A Perera1–3 1Singapore National Eye Centre, Singapore; 2Singapore Eye Research Institute, Singapore; 3Duke NUS Graduate Medical School, Singapore Purpose: Comparison of the demographic, ocular, systemic and microbiological characteristics of eyes with bleb related infection (BRI) and bleb related endophthalmitis (BRE). Methods: Retrospective chart review of patients with BRI from January 1996–July 2013. Identification done via the center’s longstanding endophthalmitis audit, BRI audit and laboratory database identifying all conjunctival swabs from blebs. Blebitis was defined as anterior segment inflammation with mucopurulent material in or around the bleb, with anterior chamber cells but no hypopyon. BRE was defined by the presence of hypopyon or vitreous inflammation. Results: Twenty-nine patients with blebitis and 10 with BRE were identified. Mean age of subjects (n=39) was 68.4 (±13.3) with a preponderance of men (74.4%) and Chinese ethnicity (74.4%). BRE patients were 10.7 years older than blebitis patients (P=0.026). 28 (71.8%) subjects had primary open angle glaucoma. The presenting intraocular pressure (IOP) dropped in blebitis but almost doubled in BRE (P=0.011) compared to average preinfective IOP. Two weeks after treatment, IOPs in both groups returned to close to preinfective levels. Subjects with blebitis more often had an avascular bleb (88.0%) while those with BRE trended toward a moderately vascular bleb (50%). The distribution of causative microorganisms between the groups was similar. Conclusion: Our study indicates that risk factors are similar in both groups even though the visual outcome and clinical course, in the form of IOP findings and bleb vascularity, can diverge significantly. The decreased IOP in blebitis subjects represents objective evidence of subclinical leaks or bleb sweating. Keywords: bleb related endophthalmitis, blebitis, post-trabeculectomy

Clarissa Shu Ming Cheng,1,2 Yi Fang Lee,2 Charles Ong,3 Zhu Li Yap,2 Andrew Tsai,2 Aditi Mohla,2 Monisha E Nongpiur,4 Tin Aung,2,4 Shamira A Perera2,4 1Department of Ophthalmology, Tan Tock Seng Hospital, 2Department of Ophthalmology, Singapore National Eye Centre, 3Yong Loo Lin School of Medicine, National University of Singapore, 4Singapore Eye Research Institute, Singapore National Eye Centre, Singapore Background: To compare retinal vessel oxygenation and vessel caliber in primary angle-closure glaucoma (PACG), primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and normal controls, as well as between eyes of asymmetrical glaucoma severity.Methods: This was a prospective, cross-sectional study. The 159 subjects (PACG, n=39; POAG, n=41; NTG, n=41; normal controls, n=38) underwent retinal oxygen saturation measurements using the Oxymap T1 Retinal Oximeter, optical coherence tomography, and Humphrey visual field testing. Retinal oxygen saturation and vessel diameter were compared between the glaucoma groups and normal controls, as well as between eyes of asymmetrical glaucoma severity. Kruskal–Wallis test was performed for comparison among different subtypes of glaucoma. Wilcoxon signed-rank test was used to compare the inter-eye differences.Results: Compared to normal controls, arteriolar oxygen saturation was increased in PACG eyes (P=0.048) but not in POAG or NTG eyes. There were no significant differences in oxygen saturation in venules or arteriovenous (AV) difference in all three glaucoma groups. Venular diameter was significantly reduced in all glaucoma groups compared to normal controls (P

Genome editing in non-model bacteria is important to understand gene-to-function links that may differ from those of model microorganisms. Although species of the Burkholderia cepacia complex (Bcc) have great biotechnological capacities, the limited genetic tools available to understand and mitigate their pathogenic potential hamper their utilization in industrial applications. To broaden the genetic tools available for Bcc species, we developed RhaCAST, a targeted DNA insertion platform based on a CRISPR-associated transposase driven by a rhamnose-inducible promoter. We demonstrated the utility of the system for targeted insertional mutagenesis in the Bcc strains B. cenocepacia K56-2 and Burkholderia multivorans ATCC17616. We showed that the RhaCAST system can be used for loss- and gain-of-function applications. Importantly, the selection marker could be excised and reused to allow iterative genetic manipulation. The RhaCAST system is faster, easier, and more adaptable than previous insertional mutagenesis tools available for Bcc species and may be used to disrupt pathogenicity elements and insert relevant genetic modules, enabling Bcc biotechnological applications., Importance: Species of the Burkholderia cepacia complex (Bcc) have great biotechnological potential but are also opportunistic pathogens. Genetic manipulation of Bcc species is necessary to understand gene-to-function links. However, limited genetic tools are available to manipulate Bcc, hindering our understanding of their pathogenic traits and their potential in biotechnological applications. We developed a genetic tool based on CRISPR-associated transposase to increase the genetic tools available for Bcc species. The genetic tool we developed in this study can be used for loss and gain of function in Bcc species. The significance of our work is in expanding currently available tools to manipulate Bcc., Competing Interests: The authors declare no conflict of interest.

We report here the complete annotated closed genome sequence of Burkholderia vietnamiensis LMG16232. There were three contigs, with a combined size of 6,739,172 bp and a GC content of 67%., Competing Interests: The authors declare no conflict of interest.

Purpose: To determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS)., Methods: Nine New Zealand white rabbits were subjected to MIGS with intraoperative implantation of the PreserFlo MicroShunt. Rabbits were then administered with subconjunctival injections of phosphate buffered saline (PBS) (n=4) or with VPA (n=5). Bleb morphology was examined by slit-lamp biomicroscopy and in vivo confocal microscopy. Postoperative day 28 tissues were examined by immunohistochemical evaluation and label-free multiphoton microscopy to visualise the collagen matrix, by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay and immunofluorescent labelling for Ki67 expression to detect apoptosis and cell growth, and by real-time quantitative PCR to measure Col1a1 , Fn , and Smad6 transcript expression., Results: VPA-treated blebs were detectable on day 28, while the PBS-treated blebs were not detectable by day 14. VPA-treated blebs were diffuse, extended posteriorly with near normal conjunctival vascularity and featured a combination of reticular/blurred stromal pattern with evidence of relatively large stromal cysts. Instead of the deposition of thick, disorganised collagen fibres characteristic of the PBS bleb, the VPA bleb contained conspicuously thinner collagen fibres which were associated with similarly thinner fibronectin fibres. In corroboration, Col1a1 and Fn mRNA expression was reduced in the VPA blebs, while increased Smad6 expression implicated the disruption of the transforming growth factor beta pathway. Apoptosis and cell growth profiles appeared similar with both treatments., Conclusions: The results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background Context: Low back pain is a major cause of morbidity and disability worldwide and is responsible for vast societal impact. Rates of surgical intervention for lumbar spine disorders continue to rise but poor outcomes remain common. Understanding how the social determinants of health (SDH) influence spinal surgical outcomes stands to inform appropriately tailored care practices and lead to better patient outcomes., Purpose: To determine the relationships between the SDH and pain, opioid use, disability and work absenteeism following lumbar spine surgery., Study Design: Systematic review and narrative synthesis., Methods: We searched Embase, the Cochrane Library, Medline, and Web of Science from inception to April 21, 2020. Studies eligible for inclusion involved participants receiving lumbar spine surgery and investigated the relationship between at least one SDH and post-surgical pain, opioid use, disability or work absenteeism. We evaluated the risk of bias of included studies and used the PROGRESS-Plus framework to organize a narrative synthesis of findings., Results: Relevant data was extracted from twenty-three studies involving 30,987 adults from 12 countries. A total of 107 relationships between the SDH and post-surgical outcomes were evaluated, 67 in multivariate analyses. Education was investigated in 23 analyses (14 studies): 70% revealed significant independent relationships between lower education and poorer outcomes. Socioeconomic status was investigated in nine analyses (four studies): 67% revealed independent relationships between lower socioeconomic status and poorer outcomes. Gender was investigated in 40 analyses (22 studies): indications that male versus female sex was associated with poorer outcomes were equivocal. Place of residence, race/ethnicity, and social capital were infrequently investigated., Conclusions: Low educational attainment and low-income status are clear independent contributors to poorer outcomes following lumbar spine surgery. Occupational factors and work context are likely to be influential. Further research is critical to guide best-practice spinal surgery through a health equity lens., Study Registration: PROSPERO registration number CRD42015015778., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This study aimed to compare the effectiveness of combination therapy consisting of low-dose mitomycin C (MMC) and valproic acid (VPA) against high-dose MMC for improving the scar phenotype in minimally invasive glaucoma surgery (MIGS)., Methods: A rabbit model of MIGS incorporating the PreserFlo MicroShunt was treated with high (0.4 mg/mL) or low (0.1 mg/mL) doses of MMC or with combination therapy consisting of low-dose (0.1 mg/mL) MMC and VPA. Operated eyes were examined by live ocular imaging, histochemical evaluation, multiphoton quantitation of collagen characteristics, and molecular analyses., Results: Although high-dose MMC obliterated the vasculature, combination therapy vastly improved the postoperative tissue morphology by maintaining the vasculature without increased vascularization. Combination therapy also altered collagen morphology and reduced encapsulation of the MicroShunt distal end, which remained at risk with MMC treatment alone. Multiphoton quantitation indicated that the combination therapy significantly reduced collagen density and fiber dimensions compared with monotherapy. At the molecular level, combination therapy significantly reduced Vegfa, Vegfc, and Vegfd expression and inhibited Col1a1 upregulation from baseline levels, all of which low-dose MMC alone was unable to achieve. Notably, COL1A1 protein levels appeared more consistently suppressed by combination therapy compared with high-dose MMC alone., Conclusions: Compared with high-dose MMC, combination therapy was less toxic by sparing the vasculature and potentially more effective in reducing scarring via the regulation of collagen content and organization., Translational Relevance: VPA may be combined with low-dose MMC to replace high-dose MMC to deliver safe and effective anti-scarring outcomes.

Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei , Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have antimicrobial properties that warrant further investigations to determine their therapeutic potential against Burkholderia infections.

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.

Burkholderia species have environmental, industrial and medical significance, and are important opportunistic pathogens in individuals with cystic fibrosis (CF). Using a combination of existing and newly determined genome sequences, this study investigated prophage carriage across the species B. vietnamiensis , and also isolated spontaneously inducible prophages from a reference strain, G4. Eighty-one B. vietnamiensis genomes were bioinformatically screened for prophages using PHASTER (Phage Search Tool Enhanced Release) and prophage regions were found to comprise up to 3.4% of total genetic material. Overall, 115 intact prophages were identified and there was evidence of polylysogeny in 32 strains. A novel, inducible Mu-like phage (vB_BvM-G4P1) was isolated from B. vietnamiensis G4 that had lytic activity against strains of five Burkholderia species prevalent in CF infections, including the Boston epidemic B. dolosa strain SLC6. The cognate prophage to vB_BvM-G4P1 was identified in the lysogen genome and was almost identical (>93.5% tblastx identity) to prophages found in 13 other B. vietnamiensis strains (17% of the strain collection). Phylogenomic analysis determined that the G4P1-like prophages were widely distributed across the population structure of B. vietnamiensis . This study highlights how genomic characterization of Burkholderia prophages can lead to the discovery of novel bacteriophages with potential therapeutic or biotechnological applications., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Purpose: Although some studies suggest a strong link between retinal vessel oxygenation and damage to the retinal nerve fiber layer (RNFL) seen in glaucoma, it has yet to be conclusively proven. This study compares intraocular retinal oximetry in glaucomatous eyes displaying asymmetrically affected hemifields across different subgroups of glaucoma, namely primary angle closure glaucoma (PACG), primary open-angle glaucoma (POAG), and normal tension glaucoma (NTG)., Methods: In this prospective cross-sectional study, 99 patients (PACG, n=28; POAG, n=37; NTG, n=34) underwent retinal oxygenation and vessel caliber measurements using the Oxymap T1 Retinal Oximeter, Cirrus optical coherence tomography, and Humphrey visual field testing. For the comparison between different subtypes of glaucoma, an analysis of variance with Bonferroni method was performed. Intraeye differences were compared with a paired t test. Determination of the more affected and less affected hemifield was made using the Humphrey visual field pattern deviation plot., Results: Considering the mean deviation and Advanced Glaucoma Interventional Study score, the visual field defects were milder in NTG as compared with POAG and PACG (P<0.05).Arteriole diameter was smaller in the more affected hemifield compared with the less affected hemifield in patients with PACG (109.30±18.07 vs. 120.57±17.92; P=0.023) and NTG (109.36±13.79 vs. 117.46±17.72; P=0.04). The more affected hemifield had a significantly thinner RNFL than the less affected hemifield in patients across all 3 groups, though this was only significant in PACG (P=0.02) and NTG patients (P<0.01). In all 3 groups, although the less affected hemifield tended to have a marginally higher arteriole and venule oxygen saturation than the more affected hemifield, no statistical significance was reached. There were no significant differences in arteriovenous difference between the more and less affected hemifield in all 3 groups., Conclusions: In our study, localized visual field losses were not associated with changes in retinal oximetry but were associated with narrower retinal arteriolar diameters in PACG and NTG. The RNFL was thinner in the more affected hemifield in these 2 groups but this was not so marked in the POAG sample, possibly limiting our ability to find a difference in arteriolar diameter there.

Thrombophilia has recently been reported to be increased in patients with cystic fibrosis (CF). We wanted to determine whether this was applicable to our population with CF and how our patients compared to the previously reported groups. Seventy one pediatric CF patients were assessed for a thrombophilic tendency, using a lupus anticoagulant screen, protein C, protein S, antithrombin assay, and activated protein C resistance (APCR) screen. The incidence of activate protein C resistance (4.2%) was within expected limits for the general population as was the incidence of antithrombin deficiency. However there was a marked increase in the incidence of lupus anticoagulants (18%) and 14% and 19.7% of the patients showed a reduced protein C and protein S, respectively, far in excess of the general population. This increased incidence of thrombophilia was not related to any specific CF phenotype and while perturbed liver function cannot be entirely ruled out, it appeared unlikely to be responsible for all the abnormal coagulation findings. Despite the apparent thrombophilic tendency, no clinically evident thrombotic episodes were noted during the study period. Thrombophilia is of concern because of the increasingly frequent placement of indwelling catheters in CF patients. The precise cause for the thrombophilic tendency in CF patients is unknown at this stage.

Background: Pleural dissemination of pseudomyxoma peritonei (PMP) is considered an advanced/terminal disease. We review our experience with thoracic cytoreductive surgery (CRS) and intraoperative hyperthermic intrathoracic chemotherapy (HITHOC) for the treatment of pleural recurrence of PMP following previous intraabdominal surgery., Patients and Methods: An observational study of five patients with pleural dissemination from PMP treated with thoracic CRS and intraoperative HITHOC with mitomycin C for 90 min at 41.5 degrees C., Results: There were three men. The mean age was 46.5 (10.5) years. Postoperatively, one patient developed Grade I, one patient developed Grade III and one patient developed Grade IV postoperative complication. Two patients had an unremarkable postoperative recovery. Only one patient has died 38 months since treatment from abdominal complication of this disease with no evidence of thoracic disease. The four surviving patients are still alive 4.6-47.4 months after treatment. Two patients have evidence of an intraabdominal recurrence., Conclusions: Thoracic CRS and intraoperative HITHOC is a safe and effective procedure to treat pleural dissemination from PMP. Long-term disease-free survival can be achieved from this treatment for which no other potentially curative therapy has been described.