Tibial tubercle transfer leads to clinically relevant improvement in patients with patellar maltracking without instability: a systematic review and meta-analysis.

PURPOSE: To assess the different surgical techniques and their outcomes following tibial tubercle transfer (TTT) in patients with patellar maltracking. METHODS: A systematic search of the literature was performed in PubMed, EMBASE and Cochrane Library. Studies reporting patient-reported outcome measures (PROMs) or clinical outcome following: TTT in patients with patellar maltracking were included. Collected PROMs were Lysholm, Kujala, IKDC score, and VAS pain. Clinical outcome included reported clinical success, patient satisfaction, complications and removal of hardware (ROH). Overall pre-, post-operative and change scores were estimated using random-effects meta-analysis models. Results were reported as overall mean and per transfer direction. RESULTS: A total of 26 studies and 761 patients (818 knees, mean age 35 years, mean follow-up 5.0 years) were included. In 73% of the studies, surgery was performed after failed conservative treatment. Transfer direction was anteromedial in 76% of all procedures. Overall Lysholm score improved from 61 to 91, Kujala from 52 to 85, IKDC from 53 to 81, and VAS from 6.2 to 2.5, respectively. Clinical success was reported in 79% of patients, and 80% of patients reported to have satisfactory results. Rates of complications and ROH were 13% and 29%, respectively. CONCLUSIONS: TTT for management of patellar maltracking can lead to good results with clinically meaningful improvement, an overall clinical success of 79% and overall patient satisfaction of 80% when appreciating the underlying anatomic condition and using appropriate technique. The level of evidence was low, and large-scale prospective, comparative cohort studies with uniform outcome scales are needed to confirm these findings. LEVEL OF EVIDENCE: IV.

Are hospitals delivering appropriate VTE prevention? The venous thromboembolism study to assess the rate of thromboprophylaxis (VTE start).

The 7th conference of the American College of Chest Physicians (ACCP7) provides recommendations on the type, dose, and duration of thromboprophylaxis in hospitalized patients at risk of venous thromboembolism (VTE), but the extent to which hospitals follow these criteria has not been well studied. Discharge and billing records for patients admitted to any of 16 acute-care hospitals from January 2005 to December 2006 were obtained. Patients 18 years or older who had an inpatient stay >or=2 days and no apparent contraindications for thromboprophylaxis were grouped into the categories of critical care, surgery and medically ill before being assessed for additional VTE risk factors based on the diagnostic criteria outlined in ACCP7. For patients at risk, the recommended type (mechanical or pharmacologic), dose, and duration of thromboprophylaxis was identified based on the guidelines and compared to the regimen actually received, if any. Among the 258,556 hospitalized patients, 68,278 (26.4%) were determined to be at risk of VTE without apparent contraindications for thromboprophylaxis. The proportions of patients who received the appropriate type, dose, and duration of thromboprophylaxis were 10.5, 9.8, and 17.9% for critical care, medical, and surgical patients, respectively. Of those at risk, 36.8% received no thromboprophylaxis and an additional 50.2% received thromboprophylaxis deemed inappropriate for one or more reasons. The implementation of ACCP7 guidelines for type, dosage, and duration of thromboprophylaxis is low in patients at risk of VTE. There is a need for physicians and health systems to improve awareness and implementation of recommended thromboprophylaxis.

A novel programme to evaluate and communicate 10-year risk of CHD reduces predicted risk and improves patients' modifiable risk factor profile.

AIMS: We assessed whether a novel programme to evaluate/communicate predicted coronary heart disease (CHD) risk could lower patients' predicted Framingham CHD risk vs. usual care. METHODS: The Risk Evaluation and Communication Health Outcomes and Utilization Trial was a prospective, controlled, cluster-randomised trial in nine European countries, among patients at moderate cardiovascular risk. Following baseline assessments, physicians in the intervention group calculated patients' predicted CHD risk and were instructed to advise patients according to a risk evaluation/communication programme. Usual care physicians did not calculate patients' risk and provided usual care only. The primary end-point was Framingham 10-year CHD risk at 6 months with intervention vs. usual care. RESULTS: Of 1103 patients across 100 sites, 524 patients receiving intervention, and 461 receiving usual care, were analysed for efficacy. After 6 months, mean predicted risks were 12.5% with intervention, and 13.7% with usual care [odds ratio = 0.896; p = 0.001, adjusted for risk at baseline (17.2% intervention; 16.9% usual care) and other covariates]. The proportion of patients achieving both blood pressure and low-density lipoprotein cholesterol targets was significantly higher with intervention (25.4%) than usual care (14.1%; p < 0.001), and 29.3% of smokers in the intervention group quit smoking vs. 21.4% of those receiving usual care (p = 0.04). CONCLUSIONS: A physician-implemented CHD risk evaluation/communication programme improved patients' modifiable risk factor profile, and lowered predicted CHD risk compared with usual care. By combining this strategy with more intensive treatment to reduce residual modifiable risk, we believe that substantial improvements in cardiovascular disease prevention could be achieved in clinical practice.

Evidence-based interventions to improve patient compliance with antihypertensive and lipid-lowering medications.

The MEDLINE database was searched from 1972 to June 2002 to identify studies of interventions designed to improve compliance with antihypertensive or lipid-lowering medications. Studies were required to employ a controlled design, follow patients for >or=6 months and measure compliance by a method other than patient self-report. The literature review yielded 62 studies describing 79 interventions. Overall, 56% of interventions were reported to improve patient compliance. When only those studies meeting minimum criteria for methodological quality were considered, 22 interventions remained and 12 were recommended, because they demonstrated a significant improvement in compliance. Recommended interventions included fixed-dose combination drugs, once-daily or once-weekly dosing schedules, unit-dose packaging, educational counselling by telephone, case management by pharmacists, treatment in pharmacist- or nurse-operated disease management clinics, mailed refill reminders, self-monitoring, dose-tailoring, rewards and various combination strategies. Personalised, patient-focused programs that involved frequent contact with health professionals or a combination of interventions were the most effective at improving compliance. Less-intensive strategies, such as prescribing products that simplify the medication regimen or sending refill reminders, achieved smaller improvements in compliance but may be cost-effective due to their low cost.

Efficacy of transpupillary thermotherapy (TTT) in the treatment of occult subfoveal choroidal neovascularization in age-related macular degeneration.

PURPOSE: To determine the efficacy of transpupillary thermotherapy (TTT) in the treatment of occult subfoveal choroidal neovascularization in patients with age-related macular degeneration (ARMD). METHODS: We conducted a retrospective review of patients with ARMD treated with TTT from June, 1999 through July, 2000 at a retina referral practice. TTT was delivered through a slit-lamp using a modified diode laser at 810 nm wavelength and a spot size of 3 mm delivered at one location for a minimum of 60 seconds duration. Re-treatment was performed at 2-month intervals if indicated. RESULTS: 81 eyes of 77 patients were included in the study. Vision improved greater than one line Snellen in 18 eyes (22%), vision was stable within one line Snellen in 38 (47%), and worsened greater than one line Snellen in 25 (31%). Patients had a mean follow-up of 9 months. The average number of treatments was 1.37 (range 1 to 4). Pretreatment vision was less than or equal to 20/200 in 54% of eyes. CONCLUSIONS: Transpupillary thermotherapy may stabilize visual acuity in a majority of patients with occult subfoveal choroidal neovascularization secondary to ARMD. Proof of therapeutic benefit is best determined by a randomized clinical trial that is currently underway (TTT4CNV).

Macular translocation with radial scleral ouffolding: experimental studies and initial human results.

PURPOSE: Different techniques have been proposed for translocating the macula in patients with subfoveal neovascularization secondary to age-related macular degeneration. A new approach utilizing radial outfolding of the sclera was investigated. MATERIALS AND METHODS: Surgical techniques and retinal displacement were evaluated in animal trials using metal scleral clips. Successful translocation and reattachment of the retina was achieved in eight rabbits (eight eyes). We conducted a retrospective review of macular translocation surgery, performed with radial scleral outfolding, in a series of five consecutive human patients (five eyes) using full-thickness transscleral mattress sutures. RESULTS: After surgery, vision improved in two of five patients, with one patient achieving a visual acuity of 20/50. The mean angle of rotation was 11.5 deg (range 8.6 -15.1). The mean amount of foveal displacement was 1,276 pm (range 852-1,620). Complications included one case of retinal detachment, one of diplopia, and one of subretinal hemorrhage. CONCLUSIONS: Limited macular translocation by radial scleral outfolding can improve vision in selected patients. Radial evagination appears to be as effective as circumferential infolding.

Transcorneal oxygen therapy for glaucoma associated with sickle cell hyphema.

PURPOSE: To study three patients with glaucoma caused by sickle cell hyphema who were successfully treated with transcorneal oxygen therapy. METHODS: Case reports. Three patients with increased intraocular pressure caused by sickle cell hyphema were administered transcorneal oxygen therapy using humidified oxygen at a flow rate that ranged from 1 to 3 l/minute. RESULTS: All three patients had a dramatic reduction in their intraocular pressure within hours of receiving oxygen therapy. No complications were associated with the oxygen therapy. CONCLUSION: Transcorneal oxygen therapy can reduce intraocular pressure in patients with glaucoma from sickle cell hyphema. Further study is warranted to evaluate this new therapy.

An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile.

Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.

Protein splicing in the absence of an intein penultimate histidine.

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.

Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage.

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.

The cyclization and polymerization of bacterially expressed proteins using modified self-splicing inteins.

Mini-inteins derived from Synechocystis sp. (Ssp DnaB intein) and Mycobacterium xenopi (Mxe GyrA intein) that have been modified to cleave peptide bonds at their C and N termini, respectively, were cloned in-frame to the N and C termini of a target protein. Peptide bond cleavage of the modified inteins generated an N-terminal cysteine and a C-terminal thioester on the same protein. These complementary reactive groups underwent intra- or intermolecular condensation to generate circular or polymeric protein species with a new peptide bond at the site of ligation. Three cyclic peptides, BBP, an organ specific localization peptide; RGD, an inhibitor of platelet aggregation; and CDR-H3/C2, which inhibits HIV-1 replication, were isolated using the two-intein system. BBP, RGD, and CDR-H3/C2 had masses of 977.1, 1119.9, and 2098.6 g/mol, respectively, as determined by matrix-assisted laser desorption-time of flight mass spectrometry, which agreed well with the values of 977.2, 1120.3, and 2098.3 g/mol, respectively, predicted for the cyclic species. This system was used to cyclize proteins as large as 395 amino acids. Furthermore, multimers of thioredoxin were formed upon concentration of the reactive species, indicating the potential to form novel biomaterials based on fibrous proteins.

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation.

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.

The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum.

The smallest known intein, found in the ribonucleoside diphosphate reductase gene of Methanobacterium thermoautotrophicum (Mth RIR1 intein), was found to splice poorly in Escherichia coli with the naturally occurring proline residue adjacent to the N-terminal cysteine of the intein. Splicing proficiency increased when this proline was replaced with an alanine residue. However, constructs that displayed efficient N- and C-terminal cleavage were created by replacing either the C-terminal asparagine or N-terminal cysteine of the intein, respectively, with an alanine. Furthermore, these constructs were used to specifically generate complementary reactive groups on protein sequences for use in ligation reactions. Reaction between an intein-generated C-terminal thioester on E. coli maltose-binding protein (43 kDa) and an intein-generated cysteine at the N terminus of either T4 DNA ligase (56 kDa) or thioredoxin (12 kDa) resulted in the ligation of the proteins through a native peptide bond. Thus the smallest of the known inteins is capable of splicing and its unique properties extend the utility of intein-mediated protein ligation to include the in vitro fusion of large, bacterially expressed proteins.

Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli.

The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.

The effect of silicone ocular surgical devices on serum IgG binding to silicones.

PURPOSE: To determine whether silicone materials used in retinal detachment repair and cataract surgery increase serum IgG binding to silicone and identify correlations with complications of ocular surgery. METHODS: Serum from 49 patients who had ocular surgery using silicone materials was examined. Patient groups included scleral buckling (n = 25), silicone oil tamponade (n = 3), scleral buckling and silicone oil tamponade (n = 9), and silicone lens implants after cataract extraction (n = 12). Convalescent samples for all patients and preoperative samples from 19 patients (18 scleral buckling and one silicone oil tamponade) were examined. Postoperative complications were monitored for up to 108 months (mean, 10.7 months; mode, 1.5 months; range, 1 to 108 months). Samples were evaluated for the extent of IgG binding to silicones using a micromodification of a previously described enzyme-linked immunosorbent assay method. RESULTS: In 19 patients, IgG binding levels in preoperative samples were 21 arbitrary units (AU) or less. Of the 25 buckling patients, one developed complications; however, in all patients the postoperative levels of IgG binding to silicone were low (2.2 to 20.0 AU). Although four silicone lens patients developed mild complications, none displayed postoperative IgG binding levels of greater than 20 AU. Three patients who underwent both scleral buckling and silicone oil tamponade developed complications; one of these patients, who was also noted to have systemic connective tissue disease, had a significant elevation in postoperative serum IgG binding to silicone. CONCLUSIONS: Statistically significant elevations of serum IgG binding to silicone were noted postoperatively in only one patient who had a systemic connective tissue disease. The complication rate and frequency of enhanced serum IgG binding to silicone was low, making correlations to surgical complications difficult. Examination of matched samples suggested that if ocular exposure to silicone implants enhances the level of serum IgG binding to silicones, it must be a rare event that should not alter the clinical use of these important devices.