A method for rapid and reliable quantification of VEGF-cell binding activity.

Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.

Anti-Scg3 Gene Therapy to Treat Choroidal Neovascularization in Mice.

Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. The disease is currently treated with anti-angiogenic biologics, including aflibercept, against vascular endothelial growth factor (VEGF) but with limited efficacy, treatment resistance and requirement for frequent intravitreal injections. Although anti-VEGF gene therapy may provide sustained therapy that obviates multiple injections, the efficacy and side effects related to VEGF pathway targeting remain, and alternative strategies to block angiogenesis independently of VEGF are needed. We recently reported that secretogranin III (Scg3) induces only pathological angiogenesis through VEGF-independent pathways, and Scg3-neutralizing antibodies selectively inhibit pathological but not physiological angiogenesis in mouse proliferative retinopathy models. Anti-Scg3 antibodies synergize dose-dependently with VEGF inhibitors in a CNV model. Here, we report that an adeno-associated virus-8 (AAV8) vector expressing anti-Scg3 Fab ameliorated CNV with an efficacy similar to that of AAV-aflibercept in a mouse model. This study is the first to test an anti-angiogenic gene therapy protocol that selectively targets pathological angiogenesis via a VEGF-independent mechanism. The findings support further safety/efficacy studies of anti-Scg3 gene therapy as monotherapy or combined with anti-VEGF to treat nAMD.

Secretogranin III Selectively Promotes Vascular Leakage in the Deep Vascular Plexus of Diabetic Retinopathy.

Diabetic retinopathy (DR), a leading cause of vision loss in working-age adults, induces mosaic patterns of vasculopathy that may be associated with spatial heterogeneity of intraretinal endothelial cells. We recently reported that secretogranin III (Scg3), a neuron-derived angiogenic and vascular leakage factor, selectively binds retinal vessels of diabetic but not healthy mice. Here, we investigated endothelial heterogeneity of three retinal vascular plexuses in DR pathogenesis and the therapeutic implications. Our unique in vivo ligand binding assay detected a 22.7-fold increase in Scg3 binding to retinal vessels of diabetic mice relative to healthy mice. Functional immunohistochemistry revealed that Scg3 predominantly binds to the DR-stressed CD31- deep retinal vascular plexus but not to the relatively healthy CD31+ superficial and intermediate plexuses within the same diabetic retina. In contrast, VEGF bound to healthy and diabetic retinal vessels indiscriminately with low activity. FITC-dextran assays indicated that selectively increased retinal vascular leakage coincides with Scg3 binding in diabetic mice that was independent of VEGF, whereas VEGF-induced leakage did not distinguish between diabetic and healthy mice. Dose-response curves showed that the anti-Scg3 humanized antibody (hAb) and anti-VEGF aflibercept alleviated DR leakage with equivalent efficacies, and that the combination acted synergistically. These findings suggest: (i) the deep plexus is highly sensitive to DR; (ii) Scg3 binding to the DR deep plexus coincides with the loss of CD31 and compromised endothelial junctions; (iii) anti-Scg3 hAb alleviates vascular leakage by selectively targeting the DR-stressed deep plexus within the same diabetic retina; (iv) combined anti-Scg3 and anti-VEGF treatments synergistically ameliorate DR through distinct mechanisms.

Dew-Cloud-Based Hierarchical Federated Learning for Intrusion Detection in IoMT.

The coronavirus pandemic has overburdened medical institutions, forcing physicians to diagnose and treat their patients remotely. Moreover, COVID-19 has made humans more conscious about their health, resulting in the extensive purchase of IoT-enabled medical devices. The rapid boom in the market worth of the internet of medical things (IoMT) captured cyber attackers' attention. Like health, medical data is also sensitive and worth a lot on the dark web. Despite the fact that the patient's health details have not been protected appropriately, letting the trespassers exploit them. The system administrator is unable to fortify security measures due to the limited storage capacity and computation power of the resource-constrained network devices'. Although various supervised and unsupervised machine learning algorithms have been developed to identify anomalies, the primary undertaking is to explore the swift progressing malicious attacks before they deteriorate the wellness system's integrity. In this paper, a Dew-Cloud based model is designed to enable hierarchical federated learning (HFL). The proposed Dew-Cloud model provides a higher level of data privacy with greater availability of IoMT critical application(s). The hierarchical long-term memory (HLSTM) model is deployed at distributed Dew servers with a backend supported by cloud computing. Data pre-processing feature helps the proposed model achieve high training accuracy (99.31%) with minimum training loss (0.034). The experiment results demonstrate that the proposed HFL-HLSTM model is superior to existing schemes in terms of performance metrics such as accuracy, precision, recall, and f-score.

Factors associated with differential COVID-19 mortality rates in the SEAR nations: a narrative review.

Objectives: Since December 2019, the world has been grappling with the COVID-19 pandemic, which has caused severe loss of lives, the breakdown of health infrastructure, and disruption of the global economy. There is growing evidence on mortality patterns in high-income countries. However, similar evidence from low/middle-income nations is lacking. Our review aimed to describe COVID-19 mortality patterns in the WHO-SEAR nations, and explore the associated factors in order to explain such trends. Methods: A systematic and comprehensive search was undertaken in PubMed and Google Scholar to obtain maximum hits on COVID-19 mortality and its determinants in the SEAR, using a combination of MeSH terms and Boolean operators. The data were narratively synthesized in detail under appropriate themes. Results: Our search identified 6411 unique records. Mortality patterns were described in terms of important demographical and epidemiological indicators. Gaps in available evidence and paucity of adequate research in this area were also highlighted. Conclusions: This review examined significant contributors to COVID-19 mortality across SEAR nations, while emphasizing issues relating to insufficient studies and data quality, and reporting challenges and other concerns in resource-constrained settings. There is a compelling need for more work in this area, to help inform decision making and improve public-health response.

Selectively targeting disease-restricted secretogranin III to alleviate choroidal neovascularization.

Choroidal neovascularization (CNV), a leading cause of blindness in the elderly, is routinely treated with vascular endothelial growth factor (VEGF) inhibitors that have limited efficacy and potentially adverse side effects. An unmet clinical need is to develop novel therapies against other angiogenic factors for alternative or combination treatment to improve efficacy and safety. We recently described secretogranin III (Scg3) as a disease-selective angiogenic factor, causally linked to diabetic retinopathy and acting independently of the VEGF pathway. An important question is whether such a disease-selective Scg3 pathway contributes to other states of pathological angiogenesis beyond diabetic retinopathy. By applying a novel in vivo endothelial ligand binding assay, we found that the binding of Scg3 to CNV vessels in live mice was markedly increased over background binding to healthy choriocapillaris and blocked by an Scg3-neutralizing antibody, whereas VEGF showed no such differential binding. Intravitreal injection of anti-Scg3 humanized antibody Fab (hFab) inhibited Matrigel-induced CNV with similar efficacy to the anti-VEGF drug aflibercept. Importantly, a combination of anti-Scg3 hFab and aflibercept synergistically alleviated CNV. Homozygous deletion of the Scg3 gene markedly reduced CNV severity and abolished the therapeutic activity of anti-Scg3 hFab, but not aflibercept, suggesting a role for Scg3 in VEGF-independent CNV pathogenesis and therapy. Our work demonstrates the stringent disease selectivity of Scg3 binding and positions anti-Scg3 hFab as a next-generation disease-targeted anti-angiogenic therapy for CNV.

Monitoring Pathogenic Viable E. coli O157:H7 in Food Matrices Based on the Detection of RNA Using Isothermal Amplification and a Paper-Based Platform.

In recent years, the number of product recalls and contamination incidents involving pathogenic bacteria has significantly increased, and the ensuing infections continue to be an ongoing problem for public health and agriculture. Due to the widespread impact of these pathogens, there is a critical need for rapid, on-site assays that can provide rapid results. In this work, we demonstrate the development of a rapid and simple test based on the combination of reverse transcription with recombinase polymerase amplification followed by lateral flow strip detection of viable Escherichia coli O157:H7 cells by detecting the RNA of the pathogen. The optimized method can be performed for approximately 2 h with a detection limit of 10 CFU/mL of E. coli O157:H7 in buffer, spinach, and ground beef samples. Our assay is sensitive, detecting only E. coli O157:H7 and not nonpathogenic E. coli or other similar pathogens. This strategy was able to distinguish viable from nonviable bacteria and more significantly was able to detect viable but nonculturable bacteria, which is a major issue when using culture-based methods for monitoring pathogenic bacteria. An important advantage of this test is that it can provide timely identification and removal of contaminated consumables prior to distribution without an extensive sample preparation.

Peptide-Functionalized Dendrimer Nanocarriers for Targeted Microdystrophin Gene Delivery.

Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer-DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier-peptide-DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

Bioluminescent Protein-Inhibitor Pair in the Design of a Molecular Aptamer Beacon Biosensing System.

Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ (IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γ and provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems.

Levels of TGF-ß1 in peri-miniscrew implant crevicular fluid.

The peri-miniscrew implant crevicular fluid is analogous to gingival crevicular fluid, and its contents reflect the state of inflammation and health during the life of the miniscrews in the mouth. The stability of MSI is fundamental to its role as an anchorage. This study aimed to evaluate transforming growth factor-beta one (TGF-ß1) of the peri-miniscrew implant crevicular fluid (PMICF), on implant insertion, pre- and post-loading of MSIs to find a clue to their role in the stability of MSI. Fifty-two MSIs sites were placed in the mouths of 13 patients aged 12-26 years undergoing orthodontic treatment. PMICF was collected using micro-pipettes at T1 (day 0, 1 h after MSI implantation), T2 (day 1), T3/baseline (day 21, preloading of MSI), T4 (day 21, 1 h post loading), T5 (day 22, 1 day post loading), T6 (day 43, 3 weeks post loading). The levels of TGF-ß1 were estimated by enzyme-linked immunosorbent assay (ELISA). The data were subjected to statistical analysis. Of the 52 MSIs, 20 MSIs failed at T3. In the case of successful MSIs, the TGF-ß1 levels were found to monotonously decrease from T1 (~1400 pg/mL) until T3 (~700 pg/mL) and saturate thereafter. In the case of failed MSIs, the levels of TGF-ß1 at various time periods were approximately constant and of much lower value than corresponding time periods of successful MSIs. This study highlights the role of TGF- ß1 in bone metabolism around miniscrew reflecting the state of inflammation from 1 h post-implantation.

Simultaneous and high sensitive detection of Salmonella typhi and Salmonella paratyphi a in human clinical blood samples using an affordable and portable device.

Enteric fever is one of the leading causes of infection and subsequent fatality (greater than 1.8 million) (WHO 2018), especially in the developing countries due to contaminated water and food inter twinned with unhygienic practices. Clinical gold standard technique of culture-based method followed by biochemical tests demand 72+ hours for diagnosis while newly developed techniques (like PCR, RT-PCR, DNA microarray etc.) suffer from high limit of detection or involve high-cost infrastructure or both. In this work, a quick and highly specific method, SMOL was established for simultaneous detection of Salmonella paratyphi A and Salmonella typhi in clinical blood samples. SMOL consists of (i) pre-concentration of S. typhi and S. paratyphi A cells using magnetic nanoparticles followed by (ii) cell lysis and DNA extraction (iii) amplification of select nucleic acids by LAMP technique and (iv) detection of amplified nucleic acids using an affordable portable device (costs less than $70). To identify the viability of target cells at lower concentrations, the samples were processed at two different time periods of t = 0 and t = 4 h. Primers specific for the SPA2539 gene in S. paratyphi A and STY2879 gene in S. typhi were used for LAMP. Within 6 h SMOL was able to detect positive and negative samples from 55 human clinical blood culture samples and detect the viability of the cells. The results were concordant with culture and biochemical tests as well as by qPCR. Statistical power analysis yielded 100%. SMOL results were concordant with culture and biochemical tests as well as by qPCR. The sensitive and affordable system SMOL will be effective for poor resource settings.

Correction: Highly-sensitive detection of Salmonella typhi in clinical blood samples by magnetic nanoparticle-based enrichment and in-situ measurement of isothermal amplification of nucleic acids.

[This corrects the article DOI: 10.1371/journal.pone.0194817.].

Rapid Detection Device for Salmonella typhi in Milk, Juice, Water and Calf Serum.

A limit of detection of 200 CFU/mL of Salmonella typhi spiked in various sample matrices were achieved in 30 min. The sample matrices were raw/unprocessed milk, commercially available milk, juice from packed bottles, fresh juice from carts, potable water, turbid water and calf serum. The complete protocol comprised of three steps: (a) cell lysis (b) nucleic acid amplification and (c) an in situ optical detection. The cell lysis was carried out using a simple heating based protocol, while the loop-mediated isothermal amplification of DNA was carried out by an in-house designed and fabricated system. The developed system consists of an aluminum block fitted with two cartridge heaters along with a thermocouple. The system was coupled to a light source and spectrometer for a simultaneous in situ detection. Primers specific for STY2879 gene were used to amplify the nucleic acid sequence, isolated from S. typhi cells. The protocol involves 15 min of cell lysis and DNA isolation followed by 15 min for isothermal amplification and simultaneous detection. No cross-reactivity of the primers were observed at 106 CFU/mL of Escherichia coli, Vibrio cholerae, Salmonella typhimurium, Salmonella paratyphi A, Pseudomonas aeruginosa, Bacillus cereus, Lysteria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Salmonella havana. In addition, the system was able to detect S. typhi of 200 CFU/mL in a concoction of 106 CFU/mL of E. coli, 106 CFU/mL of V. cholerae, and 106 CFU/mL of hepatocyte-derived cellular carcinoma HUH7 cells. The proposed rapid diagnostic system shows a promising future in the field of food and medical diagnostics.

Identification of p38 MAPK as a novel therapeutic target for Friedreich's ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder caused by decreased expression of frataxin, a protein that localizes to mitochondria and is critical for iron-sulfur-cluster (ISC) assembly. There are no proven effective treatments for FRDA. We previously screened a random shRNA library and identified a synthetic shRNA (gFA11) that reverses the growth defect of FRDA cells in culture. We now report that gFA11 decreases cytokine secretion in primary FRDA fibroblasts and reverts other changes associated with cell senescence. The gene-expression profile induced by gFA11 is remarkably similar to the gene-expression profile induced by the p38 MAPK inhibitor SB203580. We found that p38 phosphorylation, indicating activation of the p38 pathway, is higher in FRDA cells than in normal control cells, and that siRNA knockdown of frataxin in normal fibroblasts also increases p38 phosphorylation. Treatment of FRDA cells with p38 inhibitors recapitulates the reversal of the slow-growth phenotype induced by clone gFA11. These data highlight the involvement of the p38 MAPK pathway in the pathogenesis of FRDA and the potential use of p38 inhibitors as a treatment for FRDA.

Highly-sensitive detection of Salmonella typhi in clinical blood samples by magnetic nanoparticle-based enrichment and in-situ measurement of isothermal amplification of nucleic acids.

Enteric fever continues to be a major cause of mortality and morbidity globally, particularly in poor resource settings. Lack of rapid diagnostic assays is a major driving factor for the empirical treatment of enteric fever. In this work, a rapid and sensitive method 'Miod' 'has been developed. Miod includes a magnetic nanoparticle-based enrichment of target bacterial cells, followed by cell lysis and loop-mediated isothermal amplification (LAMP) of nucleic acids for signal augmentation along with concurrent measurement of signal via an in-situ optical detection system. To identify positive/negative enteric fever infections in clinical blood samples, the samples were processed using Miod at time = 0 hours and time = 4 hours post-incubation in blood culture media. Primers specific for the STY2879 gene were used to amplify the nucleic acids isolated from S. typhi cells. A limit of detection of 5 CFU/mL was achieved. No cross-reactivity of the primers were observed against 106 CFU/mL of common pathogenic bacterial species found in blood such as E. coli, P. aeruginosa, S. aureus, A. baumanni, E. faecalis, S. Paratyphi A and K. pneumonia. Miod was tested on 28 human clinical blood samples. The detection of both pre-and post-four-hours incubation confirmed the presence of viable S. typhi cells and allowed clinical correlation of infection. The positive and negative samples were successfully detected in less than 6 hours with 100% sensitivity and specificity.

A review of biomarkers in peri-miniscrew implant crevicular fluid (PMICF).

BACKGROUND: The temporary anchorage devices (TADs) which include miniscrew implants (MSIs) have evolved as useful armamentarium in the management of severe malocclusions and assist in complex tooth movements. Although a multitude of factors is responsible for the primary and secondary stability of miniscrew implants, contemporary research highlights the importance of biological interface of MSI with bone and soft tissue in augmenting the success of implants. The inflammation and remodeling associated with MSI insertion or loading are reflected through biomarkers in peri-miniscrew implant crevicular fluid (PMICF) which is analogous to the gingival crevicular fluid. Analysis of biomarkers in PMICF provides indicators of inflammation at the implant site, osteoclast differentiation and activation, bone resorption activity and bone turnover. The PMICF for assessment of these biomarkers can be collected non-invasively via paper strips, periopaper or micro capillary pipettes and analysed by enzyme-linked immunosorbent assay (ELISA) or immunoassays. The markers and mediators of inflammation have been previously studied in relation to orthodontic tooth movement include interleukins (IL-1ß, IL-2, IL-6 and IL-8), growth factors and other proteins like tumour necrosis factor (TNF-α), receptor activator of nuclear factor kappa-B ligand (RANKL), chondroitin sulphate (CS) and osteoprotegerin (OPG). Studies have indicated that successful and failed MSIs have different concentrations of biomarkers in PMICF. However, there is a lack of comprehensive information on this aspect of MSIs. Therefore, a detailed review was conducted on the subject. RESULTS: A literature search revealed six relevant studies: two on IL-1ß; one on IL-2, IL-6 and IL-8; one on TNF-α; one on CS; and one on RANKL/OPG ratio. One study showed an increase in IL-1ß levels upon MSI loading, peak in 24 hours (h), followed by a decrease in 21 days to reach baseline in 300 days. A 6.87% decrease in IL-2 levels was seen before loading and a 5.97% increase post-loading. IL-8 showed a 6.31% increase after loading and IL-6 increased by 3.08% before MSI loading and 15.06% after loading. RANKL/OPG ratio increased in loaded compared to unloaded MSIs. CONCLUSIONS: Cytokines (mainly ILs and TNF-α) and RANKL/OPG ratio showed alteration in PMICF levels upon loading of MSIs as direct or indirect anchorage.

Phacoemulsification versus small incision cataract surgery in patients with uveitis.

AIM: To compare the safety and efficacy of phacoemulsification and small incision cataract surgery (SICS) in patients with uveitic cataract. METHODS: In a prospective, randomized multi-centric study, consecutive patients with uveitic cataract were randomized to receive phacoemulsification or manual SICS by either of two surgeons well versed with both the techniques. A minimum inflammation free period of 3mo (defined as less than 5 cells per high power field in anterior chamber) was a pre-requisite for eligibility for surgery. Superior scleral tunnel incisions were used for both techniques. Improvement in visual acuity post-operatively was the primary outcome measure and the rate of post-operative complications and surgical time were secondary outcome measures, respectively. Means of groups were compared using t-tests. One way analysis of variance (ANOVA) was used when there were more than two groups. Chi-square tests were used for proportions. Kaplan Meyer survival analysis was done and means for survival time was estimated at 95% confidence interval (CI). A P value of <0.05 was considered statistically significant. RESULTS: One hundred and twenty-six of 139 patients (90.6%) completed the 6-month follow-up. Seven patients were lost in follow up and another six excluded due to either follow-up less than six months (n=1) or inability implant an intraocular lens (IOL) because of insufficient capsular support following posterior capsule rupture (n=5). There was significant improvement in vision after both the procedures (paired t-test; P<0.001). On first postoperative day, uncorrected distance visual acuity (UDVA) was 20/63 or better in 31 (47%) patients in Phaco group and 26 (43.3%) patients in SICS group (P=0.384). The mean surgically induced astigmatism (SIA) was 0.86±0.34 dioptres (D) in the phacoemulsification group and 1.16±0.28 D in SICS group. The difference between the groups was significant (t-test, P=0.002). At 6mo, corrected distance visual acuity (CDVA) was 20/60 or better in 60 (90.9%) patients in Phaco group and 53 (88.3%) in the manual SICS group (P=0.478). The mean surgical time was significantly shorter in the manual SICS group (10.8±2.9 versus 13.2±2.6min) (P<0.001). Oral prednisolone, 1 mg/kg body weight was given 7d prior to surgery, continued post-operatively and tapered according to the inflammatory response over 4-6wk in patients with previously documented macular edema, recurrent uveitis, chronic anterior uveitis and intermediate uveitis. Rate of complications like macular edema (Chi-square, P=0.459), persistent uveitis (Chi-square, P=0.289) and posterior capsule opacification (Chi-square, P=0.474) were comparable between both the groups. CONCLUSION: Manual SICS and phacoemulsification do not differ significantly in complication rates and final CDVA outcomes. However, manual SICS is significantly faster. It may be the preferred technique in settings where surgical volume is high and access to phacoemulsification is limited, such as in eye camps. It may also be the appropriate technique for uveitic cataract under such circumstances.

Phenotypic Screening for Friedreich Ataxia Using Random shRNA Selection.

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix and regulates the iron-sulfur cluster (ISC) assembly complex. ISCs are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain. Decreased expression of frataxin is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. In media with beta-hydroxybutyrate (BHB) as carbon source, primary FRDA fibroblasts grow poorly and/or lose viability over several days. We screened a random, short-hairpin-RNA (shRNA)-expressing library in primary FRDA fibroblasts and identified two shRNAs that reverse the growth/viability defect in BHB media. One of these two clones increases frataxin expression in primary FRDA fibroblasts, either as a vector-expressed shRNA or as a transfected short-interfering RNA (siRNA).

A randomized controlled trial of peeling and aspiration of Elschnig pearls and neodymium: yttrium-aluminium-garnet laser capsulotomy.

AIM: To compare surgical peeling and aspiration and neodymium yttrium garnet laser capsulotomy for pearl form of posterior capsule opacification (PCO). METHODS: A prospective, randomized, double blind, study was done at Rotary Eye Hospital, Maranda, Palampur, India, Santosh Medical College Hospital, Ghaziabad, India and Laser Eye Clinic, Noida India. Consecutive patients with pearl form of PCO following surgery, phacoemulsification, manual small incision cataract surgery and conventional extracapsular cataract extraction (ECCE) for age related cataract, were randomized to have peeling and aspiration or neodymium yttrium garnet laser capsulotomy. Corrected distance visual acuity (CDVA), intra-operative and post-operative complications were compared. RESULTS: A total of 634 patients participated in the study, and 314 (49.5%) patients were randomized to surgical peeling and aspiration group and 320 (50.5%) to the Nd:YAG laser group. The mean pre-procedural logMAR CDVA in peeling and neodymium: yttrium-aluminium-garnet (Nd:YAG) laser group was 0.80±0.25 and 0.86±0.22, respectively. The mean final CDVA in peeling group (0.22±0.23) was comparable to Nd:YAG group (0.24±0.28; t test, P=0.240). There was a significant improvement in vision after both the procedures (P<0.001). A slightly higher percentage of patients in Nd:YAG laser group (283/88.3%) than in peeling group (262/83.4%) had a CDVA of 0.5 (20/63) or better at 9mo (P<0.001). On the contrary, patients having CDVA worse than 1.00 (20/200) was also significantly higher in Nd:YAG laser group as compared to peeling group (25/7.7% vs 15/4.7%, respectively). On application of ANCOVA, there was less than 0.001% risk that PCO thickness and total laser energy had no effect on rate of complications in Nd:YAG laser group and less than 0.001 % risk that PCO thickness had no effect on complications in peeling group respectively. Sum of square analysis suggests that in the Nd:YAG laser group, thick PCO had a stronger impact on complications (Fischer test probability, Pr<0.0001) than thin PCO and total laser energy (Fischer test probability, Pr<0.002), respectively; similarly, in peeling group, thick PCO and preoperative vision had a stronger effect on complications than thin PCO, respectively (Fischer test probability, Pr<0.001).The rate of complications like uveitis (P=0.527) and cystoid macular edema (P=0.068), did not differ significantly between both the groups. However, intraocular pressure spikes (P=0.046) and retinal detachment (P<0.001) were significantly higher in Nd:YAG laser group as compared to peeling group. Retinal detachment was more common in patients having degenerative myopia (7/87.5%, P<0.001). Recurrence of pearls was the most common cause of reduction of vision in the peeling group (24/7.6%, P<0.001). CONCLUSION: There is no alternative to Nd:YAG laser capsulotomy for fibrous subtype of PCO. For pearl form of PCO, both techniques are comparable with regard to visual outcomes. Nd:YAG laser capsulotomy has a higher incidence of IOP spikes and retinal detachment whereas recurrence of pearls may occur after successful peeling and aspiration. When posterior capsulotomy is needed in patients with retinal degenerations, retinopathies and pre-existing retinal breaks, the clinician should be cautious about increased risks of possible complications of Nd:YAG laser capsulotomy.

Oral omega-3 fatty acids treatment in computer vision syndrome related dry eye.

PURPOSE: To assess the efficacy of dietary consumption of omega-3 fatty acids (O3FAs) on dry eye symptoms, Schirmer test, tear film break up time (TBUT) and conjunctival impression cytology (CIC) in patients with computer vision syndrome. SETTING AND DESIGN: Interventional, randomized, double blind, multi-centric study. METHODS: Four hundred and seventy eight symptomatic patients using computers for more than 3h per day for minimum 1 year were randomized into two groups: 220 patients received two capsules of omega-3 fatty acids each containing 180mg eicosapentaenoic acid (EPA) and 120mg docosahexaenoic acid (DHA) daily (O3FA group) and 236 patients received two capsules of a placebo containing olive oil daily for 3 months (placebo group). The primary outcome measure was improvement in dry eye symptoms and secondary outcome measures were improvement in Nelson grade and an increase in Schirmer and TBUT scores at 3 months. RESULTS: In the placebo group, before dietary intervention, the mean symptom score, Schirmer, TBUT and CIC scores were 7.5±2, 19.9±4.7mm, 11.5±2s and 1±0.9 respectively, and 3 months later were 6.8±2.2, 20.5±4.7mm, 12±2.2s and 0.9±0.9 respectively. In the O3FA group, these values were 8.0±2.6, 20.1±4.2mm, 11.7±1.6s and 1.2±0.8 before dietary intervention and 3.9±2.2, 21.4±4mm, 15±1.7s, 0.5±0.6 after 3 months of intervention, respectively. CONCLUSION: This study demonstrates the beneficial effect of orally administered O3FAs in alleviating dry eye symptoms, decreasing tear evaporation rate and improving Nelson grade in patients suffering from computer vision syndrome related dry eye.