Comparison of gal-lac operons in wild-type galactose-positive and -negative Streptococcus thermophilus by genomics and transcription analysis.

Streptococcus thermophilus is one of the most important homo-fermentative thermophilic bacteria, which is widely used as a starter culture in dairy industry. Both wild-type galactose-negative (Gal-) S. thermophilus AR333 and galactose-positive (Gal+) S. thermophilus S-3 in this study were isolated from Chinese traditional dairy products. Here, to access the mechanism of the difference of galactose utilization between strains AR333 and S-3, the expression of gal-lac operons was examined using real-time qPCR in the presence of different sugars, and the gene organization of gal-lac operons was characterized using comparative genomics analysis. As compared with medium containing glucose, the expression of gal-lac operons in AR333 and S-3 was significantly activated (> 5-fold) in the presence of galactose or lactose in the medium. More importantly, the expression of gal operon in S-3 was higher than that of AR333, suggesting that the strength of gal promoter in AR333 and S-3 may be different. The genomes of AR333 and S-3 were the first time sequenced to provide insight into the difference of gal-lac operons in these two strains. Comparative genomics analysis showed that gene order and individual gene size of gal-lac operons are conserved in AR333 and S-3. The DNA sequence of gal operon responsible for galactose utilization between AR333 and S-3 is almost identical except that galK promoter of S-3 possesses single base pair mutation (G to A substitution) at -9 box galK region. Moreover, the expression of red fluorescent protein can be activated by galK promoter of S-3, but cannot by galK promoter of AR333 in galactose medium, suggesting that gal operon is silent in AR333 and active in S-3 under galactose-containing medium. Overall, our results indicated that single point mutation at -9 box in the galK promoter can significantly affect the expression of gal operon and is largely responsible for the Gal+ phenotype of S. thermophilus.

Laser Peripheral Iridotomy versus Trabeculectomy as an Initial Treatment for Primary Angle-Closure Glaucoma.

PURPOSE: To compare laser peripheral iridotomy (LPI) with trabeculectomy as an initial treatment for primary angle-closure glaucoma (PACG) with peripheral anterior synechiae (PAS) ≥ 6 clock hours. METHODS: Patients were drawn from two randomized controlled trials. 38 eyes of 38 patients (PAS ≥ 6 clock hours) were treated with LPI (group 1) while 111 eyes of 111 PACG patients (PAS ≥ 6 clock hours) underwent primary trabeculectomy (group 2). All patients underwent a comprehensive ophthalmic examination at baseline and at postoperative visits and were followed up for a minimum of one year. RESULTS: Group 2 had higher baseline IOP (45.7 ± 14.8 mmHg versus 34.3 ± 14.3 mmHg) than group 1 and more clock hours of PAS (10.4 ± 1.9 versus 9.0 ± 2.2). IOPs at all postoperative visits were significantly lower in group 2 than in group 1 (p = 0.000). Five eyes in group 1 required trabeculectomy. 17 of the 38 eyes in group 1 (44.7%) required IOP-lowering medications as compared to seven of the 111 eyes in group 2 (6.3%). Cataract progression was documented in 2 eyes (5.3%) in group 1 and 16 eyes (14.4%) in group 2. CONCLUSIONS: Primary trabeculectomy for PACG (PAS ≥ 6 clock hours) is more effective than LPI in lowering IOP.

Construction of polyketide overproducing Escherichia coli strains via synthetic antisense RNAs based on in silico fluxome analysis and comparative transcriptome analysis.

Rapid assessment and optimization of the incompatible metabolic modules remain a challenge. Here, we developed a systematic approach to characterize the module interactions and improve the problematic modules during the 6-deoxyerythronolide B (6dEB) biosynthesis in E. coli. Tremendous differences in the overall trends of flux changes of various metabolic modules were firstly uncovered based on in silico fluxome analysis and comparative transcriptome analysis. Potential targets for improving 6dEB biosynthesis were identified through analyzing these discrepancies. All 25 predicted targets at modules of PP pathway and nucleotide metabolism were firstly tested for improving the 6dEB production in E. coli via synthetic antisense RNAs. Down-regulation of 18 targets genes leads to more than 20% increase in 6dEB yield. Combinatorial repression of targets with greater than 60% increase in 6dEB titer, e.g., anti-guaB/anti-zwf led to a 296.2% increase in 6dEB production (210.4 mg/L in flask) compared to the control (53.1 mg/L). This is the highest yield yet reported for polyketide heterologous biosynthesis in E. coli. This study demonstrates a strategy to enhance the yield of heterologous products in the chassis cell and indicates the effectiveness of antisense RNA for use in metabolic engineering.

Identification of novel knockout and up-regulated targets for improving isoprenoid production in E. coli.

Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10-45% increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174% in shake-flask and 81% in bioreactor fermentations with a maximum yield of 454 mg l(-1).

Can intraoperative intraocular pressure during primary trabeculectomy predict early postoperative pressure?

PURPOSE: To study the association of intraoperative intraocular pressure (IOP) at the conclusion of primary trabeculectomy with postoperative IOP on days 1, 7, and 30 and report the ability of intraoperative IOP to predict early postoperative IOP. PATIENTS AND METHODS: Prospective, observational, case series. Ninety-seven consecutive patients with primary open-angle or angle-closure glaucoma underwent primary trabeculectomy. The IOP was measured 3 minutes after the closure of scleral flap with a formed anterior chamber. Clinical details and complications were recorded over a 30-day follow-up. The patients were grouped as A (intraoperative IOP≤10.0 mm Hg), B (>10, ≤15.0 mm Hg), and C (>15 mm Hg). RESULTS: Mean age was 60.8±9.9 years. Forty (41.3%) patients were males. Eighty-two (84.5%) had primary angle-closure glaucoma. Mitomycin-c (MMC) was used in 75 (77.3%) eyes. Mean IOP in group C was significantly higher than group A at all visits (P=0.005, 0.002, and 0.05). Postoperative IOP was associated with intraoperative IOP, age, duration, and dose of MMC in univariable regressions. On multivariable analysis, the intraoperative IOP was associated with postoperative IOP's on day 1 (regression coefficient b=0.24, P=0.039, R(2)=0.24) and day 7 (b=0.47, P<0.001, R=0.42), but not on day 30 (b=0.22, P=0.065, R(2)=0.12). IOP on day 7 was predicted by -8.6+0.47×(intraoperative IOP)+0.27×age-11.7×(dose of MMC in mg/mL). Prediction for day 30=9.8+0.27×(intraoperative IOP). CONCLUSIONS: In patients undergoing uncomplicated primary trabeculectomy, intraoperative IOP is associated with and may predict early postoperative IOP. Adjusting the IOP during the operation may optimize postoperative IOP.

Daytime fluctuation of intraocular pressure in patients with primary angle-closure glaucoma after trabeculectomy.

PURPOSE: To evaluate the daytime fluctuation of intraocular pressure (IOP) in patients with primary angle-closure glaucoma (PACG) after trabeculectomy. PARTICIPANTS AND METHODS: A total of 176 patients with PACG participated in a clinical trial of trabeculectomy with or without releasable sutures. Applanation IOP was measured at 5, 7, and 10 AM, and 2, 6, and 10 PM at 3 months posttrabeculectomy. We documented the mean, peak, and trough IOPs, determined the fluctuation of daytime IOP, and explored the associations of IOP fluctuation with baseline factors. RESULTS: IOP measurements were obtained in 173 patients. The mean daytime IOP was 13.2±3.7 mm Hg; mean peak IOP 15.1±4.1 mm Hg, mean trough IOP 11.3±3.5 mm Hg, and mean fluctuation 3.8±2.1 mm Hg. Fluctuation was positively correlated with peak (r=0.528, R2=0.28, P<0.001) and mean IOP (r=0.278, R2=0.08, P<0.001), but not with the trough IOP (r=0.015, P=0.843). Fluctuation was lower with extent of bleb (0.6 mm Hg/unit increase in extent; 95% CI, 0.1-1.2 mm Hg) and in blebs with microcysts (1.1 mm Hg less fluctuation; 95% CI, 0.2-1.9 mm Hg). Fluctuation was not associated with sex, age, baseline IOP, extent of peripheral anterior synechia or number of glaucoma medications before surgery, mean deviation of the visual field, vertical cup:disc ratio, or the use of releasable sutures. CONCLUSIONS: The mean fluctuation of daytime IOP after trabeculectomy for PACG was about 4 mm Hg. The fluctuation was positively associated with higher peak and mean IOP and negatively associated with extent of bleb and presence of microcysts.

[Assess correlation between bleb morphology at long-term intraocular pressure effect in primary angle-closure glaucoma following trabeculectomy].

OBJECTIVE: To evaluate the correlation between morphologic appearance of blebs at 3 month and long-term intraocular pressure (IOP) effect in patients with primary angle-closure glaucoma (PACG) after trabeculectomy. METHODS: Multi-centered cases series. Data were collected from 176 patients aged ≥ 40 years with PACG who were participated in a randomized clinical trial that aimed at addressing the efficacy of augmented releasable sutures after trabeculectomy. The bleb morphology was graded using the Modified Indian Bleb Appearance Grading Scale (IBAGS) based on standard photos at 3 month after trabeculectomy. IOPs were measured with Goldmann applanation tonometer. The correlation between bleb components and other selected testing influencing factors and long-term IOP was tested by linear Logistic regression analysis. RESULTS: 150 patients (85.7%) completed 18 months of follow up. IOP was (15.6 ± 5.4) mm Hg at 18 month of post-operation. 135 eyes had an IOP ≤ 21 mm Hg without additional medications, 10 eyes ≥ 21 mm Hg, and the remaining 5 eyes required one or two medications to maintain normal IOP. Using IBAGS system, bleb was graded in 142 eyes as follows: H(0) in 3 eyes, H(1) in 45 eyes, H(2) in 90 eyes, and H(3) in 4 eyes, while V(0) was observed in 66 eyes, V(1-3) in 76 eyes. IOP at 18 months in bleb with microcysts was 2.77 mm Hg lower (ß = -2.77, 95%CI = -0.46 to -5.08) than those without microcysts and in bleb with non-vascular was 2.07 mm Hg lower (ß = -2.07, 95%CI = -0.15 to -3.98) than those with vascular at 3 months after surgery. IOP was significantly (ß = -1.20, 95%CI: -0.00 to -2.40) decreased by 1.2 mm Hg with 10 years of age increase (P < 0.05). CONCLUSIONS: Early filtering bleb with microcysts, vascular, and age are identified as important factors to predict long-term IOP effect in patients with PACG after trabeculectomy but not early morphological appearance of filtering bleb.