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Background: The acetabular version is crucial for hip function, and its accurate assessment is necessary for treating patients with hip disorders. Current studies reveal discrepancies in the precision of quantitative radiographic measurements versus CT measurements, but there is a lack of focused analysis on anteverted versus retroverted hips. This study aims to fill this gap by directly comparing the reliability of these two methods in assessing varied hip configurations., Questions/purposes: (1) How reliable are quantitative radiographic and CT methods in measuring the acetabular version angle? (2) Is there any difference in the reliability of acetabular version angle measurements using radiography compared with CT in anteverted and retroverted hips? (3) What is the extent of variation in acetabular version measurements when quantitative radiographic and CT methods are compared in anteverted and retroverted hips?, Methods: We searched our image archives for patients who had received both radiographs and CT scans between January 2020 and June 2022 and found 84 patients who met the criteria. From these patients, we selected those who presented with hip pain of different causes and who had no previous elective and/or hip trauma surgery, no hip dysplasia, and results from adequate radiographic examinations. Accordingly, 73% (61 of 84) of the patients were included in this study, and angle measurements were performed on both hips of these patients (122 hips). Standardized positioning was meticulously verified for all plain radiographs and CT scans utilized in the measurement process. We measured quantitative angles and assessed qualitative signs of retroversion, including crossover, posterior wall, and ischial spine findings. We considered a hip with at least one of these findings a retroverted hip, and the hips without these findings were included in the anteverted hip group. Three clinicians took measurements independently. Measurement reliability and agreement were examined using intraobserver and interobserver intraclass correlation coefficients (ICCs), with statistical analyses including paired and independent t-tests. To investigate the reliability of quantitative radiographic and CT methods, we assessed both intraobserver and interobserver agreements. To explore the reliability disparities in measuring the acetabular version via radiography and CT in anteverted and retroverted hips, we analyzed the agreement between measurements from both modalities in the hip groups. Furthermore, to evaluate the degree of variation in acetabular version measurements when comparing quantitative radiographic and CT methods in anteverted and retroverted hips, we utilized paired and independent t-tests to examine the measurement differences within these hip categories. The difference between radiographic and CT measurements was also evaluated by Bland-Altman analysis., Results: Quantitative radiographic measurements showed intraobserver and interobserver reliabilities with ICCs of 0.87 (95% CI 0.84 to 0.91) and 0.78 (95% CI 0.75 to 0.82), respectively, and CT measurements demonstrated higher reliabilities with ICCs of 0.92 (95% CI 0.90 to 0.93) and 0.91 (95% CI 0.89 to 0.92), respectively. The reliability of measuring the acetabular version in anteverted hips was moderate, with an ICC of 0.59 (95% CI 0.49 to 0.68). In contrast, retroverted hips showed an ICC of -0.41 (95% CI -1.17 to 0.08), indicating a lack of consistency between quantitative radiographic and CT measurements. Variation in measurement on plain radiographs in anteverted hips was less than that of retroverted hips (mean ± SD absolute difference between anteverted hips and retroverted hips 3° ± 3° versus 6° ± 4°; p = 0.0001), indicating greater variability in the radiographic measurement of retroverted hips. According to Bland-Altman analysis, we observed that the difference between radiographic and CT measurements was well outside the CI, especially in retroverted hips., Conclusion: Although quantitative radiographic measurement demonstrates acceptable intraobserver and interobserver reliabilities, its precision is lower than that of CT-based measurements. Specifically, quantitative radiographic methods are prone to a larger margin of error in retroverted hips. For more precise assessments of acetabular version, especially in retroverted hips, we recommend using CT measurement instead of the radiographic method., Level of Evidence: Level III, diagnostic study., Competing Interests: Each author certifies that there are no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article related to the author or any immediate family members. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Association of Bone and Joint Surgeons.)

The reconstruction of patients who possess multi morbid medical histories remains a challenge. With the ever-increasing number of patients with diabetes, infections, and trauma, there is a consistent need for promotion of soft tissue healing and a reliable substrate to assist with every aspect of soft tissue reconstruction, as well as the loss of fascial domain. Several proprietary products filled some of these needs but have failed to fulfill the needs of the clinician when faced with reconstructing multiple soft tissue systems, such as the integument and the musculoskeletal system. In this paper we discuss the use of decellularized human dermis (DermaPure ® , Tissue Regenix, Universal City, TX, USA) through which a unique human tissue processing technique (dCELL ® technology, Tissue Regenix, Universal City, TX, USA) and the creation of multiple product forms have proven to exhibit versatility in a wide range of clinical needs for successful soft tissue reconstruction. The background of human tissue processing, basic science, and early clinical studies are detailed, which has translated to the rationale for the success of this unique soft tissue substrate in orthoplastic reconstruction, which is also provided here in detail.

Objectives: During the coronavirus disease-2019 pandemic, masks have become mandatory for protection against the virus transmitted by breathing. This study examined the impact of surgical masks used daily on civilian facial skin., Materials and Methods: Moisture, elasticity, pore, melanin, acne, wrinkle, and sensitivity parameters of 83 volunteers were measured numerically using an API-100 skin analyzer and camera recordings. Numerical values were compared following the device's algorithm calibrated according to age, gender, and race. Finally, the obtained data were statistically evaluated and compared with the averages., Results: Pore, melanin, acne, and wrinkle parameters were higher without gender discrimination, whereas moisture and elasticity parameters were low. While a significant increase was observed in women for sensitivity, the increase was not statistically significant in men., Conclusion: The negative effects of long-term daily wearing of surgical masks on facial skin were statistically significant. Therefore, taking outdoor breaks during mask use, washing the face intermittently, using moisturizing and purifying cosmetic products, and anti-wrinkle effects have been proposed to reduce the possible defects., Competing Interests: Conflict of Interest: The authors have no conflicts of interest to declare.

This work presents a detailed review of the development of distributed acoustic sensors (DAS) and their newest scientific applications. It covers most areas of human activities, such as the engineering, material, and humanitarian sciences, geophysics, culture, biology, and applied mechanics. It also provides the theoretical basis for most well-known DAS techniques and unveils the features that characterize each particular group of applications. After providing a summary of research achievements, the paper develops an initial perspective of the future work and determines the most promising DAS technologies that should be improved.

Objective: The D-SPIRIT registry is designed to investigate the safety and effectiveness of dabigatran etexilate in patients with atrial fibrillation in routine clinical practice., Methods: D-SPIRIT is the first national, multicenter, prospective, observational, postmarketing registry that investigates the usage of dabigatran in real life. A total of 326 noveloral anticoagulant-eligible patients with atrial fibrillation who have been taking dabigatran etexilate therapy for stroke prevention at least 6 months from 9 different centers were enrolled into the registry. Patients were followed up for 2 years to evaluate the effectiveness and safety of the treatment. All adverse clinical events including bleeding, thromboembolic events, stroke, systemic embolism, transient ischemic attack, myocardial infarction, and all-cause death were recorded., Results: The mean age was 71.1±9.6 years, and 57.4% of the study participants were female. The mean CHA2DS2-VASc (congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke or transient ischemic attack [TIA], vascular disease, age 65-74 years, sex category) score was 3.4±1.6. The cumulative adverse clinical events rate was 6.30% per year. The rate for embolic events including TIA, ischemic stroke, and peripheral embolism was 1.26% per year. The rate for major bleeding was 2.20% per year, and the mortality rate was 0.94% per year., Conclusion: This registry obtained an important overview of the current safety and effectiveness of the dabigatran etexilate in Turkey. Our results indicate similar rates of thromboembolic and bleeding events with pivotal phase 3 trial and other real-life registries. However, rate of undertreatment usage of dabigatran etexilate in real life was found to be considerable.

Objective: The aim of this study was to determine the role of hip arthrography in the treatment decision making for children with Legg-Calvé-Perthes disease (LCPD)., Methods: A total of 47 consecutive children with LCPD (42 boys, 5 girls; mean age=7.5 years; range=6-10 years) who underwent operative treatment were included in the study. The patient demographics, physical examination findings (pain and hip range of motion [ROM]), standard anteroposterior and Löwenstein lateral hip radiographs, and hip arthrography data were retrospectively collected. The arthrographies were performed immediately before the surgery under general anesthesia. The patients were staged according to the Catterall and Herring classifications and examined in terms of head-at-risk signs before the study. Four sets of patient files were established based on the aforementioned data, with each child in a randomized and blinded order. Ten consultant pediatric orthopedic surgeons randomly assessed the patient files on 4 separate occasions (Set 1 vs Set 2 and Set 3 vs Set 4), with a minimum time interval of 4 weeks. In the first and second sets, the demographic and clinical information, including the age, gender, hip ROM, and hip radiographs, were presented. In the third and fourth sets, hip arthrography was presented in addition to the data from Set 1 and Set 2. The observers were instructed to choose the best treatment options. The percent agreement (PA) and Gwet's AC1 statistics were used to establish a relative level of agreement among the observers., Results: The mean intra-observer reliabilities ranged from fair to moderate after adding the hip arthrography data (Gwet's AC1 = 0.36 for Set 1 vs Set 2 and 0.42 for Set 3 vs Set 4). The mean PA was 56.6% (range = 29.8% to 78.7%) with a Gwet's AC1 value of 0.51 (range: 0.21 to 0.77) between Set 1 and Set 3 (moderate intra-observer reliability). The decision for the treatment strategy was changed in 43.4% of the patients. For inter-observer reliability, Gwet's AC1 was computed as 0.48 (moderate reliability). The correlation between the intra-observer reliability and stage progression was not significant (p>0.05) for any of the subgroups. Thus, there is a negative correlation with the disease progression., Conclusion: Hip arthrography seems to have a significant role in the treatment decision making for children with LCPD, especially in the advanced stages of the disease., Level of Evidence: Level IV, Therapeutic study.

Type-I fiber Bragg gratings photo-inscribed in hydrogen-loaded B/Ge co-doped silica single-mode optical fibers have been regenerated efficiently at 450°C, which is the lowest temperature reported so far. The mechanical strength of the annealed fiber is preserved while ensuring temperature sensing of the regenerated gratings up to 900°C. Unlike low temperature cycles (≤600°C), an annealing process at higher temperatures revealed faster regeneration for strong gratings. Changes in grating strength were also measured before the regeneration cycle. These behaviors suggest the contribution of different mechanisms to the regeneration process with different relative dynamics.

European Medicines Agency (EMA) issued a final decision on September 01, 2014 that restricts the maximum daily dose of domperidone to 30 mg and treatment duration to 7 days. This paper presents a critical review of the scientific basis of the literatures having a role in the decision of EMA on domperidone with an approach based on statistical and epidemiological perspective. Although observational studies used by EMA were published, the EMA didn't use an algorithm including "randomized clinical trials" according to evidence-based medicine when presenting their results. In conclusion, the results obtained from published studies are controversial, especially for the bias. From these publications, it cannot be concluded that domperidone exposure definitely increases the risk of "sudden cardiac death", "death associated with ventricular arrhythmia" or "ventricular arrhythmia" The most concrete result of these studies is that the risk is higher with metoclopramide exposure compared to domperidone exposure.

Background: Collagen-based synthetic bone grafts which contain tricalcium phosphate (TCP) and hydroxyapatite (HA), and collagen-based synthetic bone grafts containing only TCP have some advantages compared to autografts. Therefore, these grafts are frequently used to fill bone defects and pseudoarthrosis., Objectives: The aim of this study was to evaluate and compare the clinical, radiological and histopathological effects of TCP-HA and TCP alone + Type-1 collagen in healing lower extremity fractures in a pseudoarthrosis model in rat femurs., Material and Methods: A total of 36 female Wistar rats were randomly separated into 4 groups. Group 1 (n = 10) was the control group. A femur pseudoarthrosis model was created in Groups 2, 3 and 4. On the 90th day after the 1st surgery in Group 2 (n = 10), TCP-HA + Type-1 collagen was applied, in Group 3 (n = 10), TCP alone + type-1 collagen was applied, and in Group 4 (n = 6, the placebo group), saline solution was applied. Fixation was performed with an intramedullar pin. After 60 days and clinical and radiological scoring, all animals were sacrificed and a histopathological evaluation of the pseudoarthrosis areas was conducted., Results: In all the clinical, radiological and histopathological measurements used in the evaluations of the differences between the groups, a higher rate of union was determined in Group 2 (TCP-HA). No significant difference was determined between Group 3 and Group 4 in terms of union rates., Conclusions: The clinical, radiological and histopathological results of this study showed that TCP alone was less effective than TCP-HA in the union of a femur pseudoarthrosis model in rats. The reason for this difference was considered to be hydroxyapatite (HA).

Purpose: The two-tunnel coracoclavicular ligament reconstruction (CLR) technique is one of the treatment approaches commonly used in the surgical treatment of acromioclavicular (AC) injuries. Clavicular tunnel malposition is one of the major causes of failure in coracoclavicular ligament reconstruction. The main purpose of this study was to investigate the effects of clavicular tunnel placement on tendon loading in the CLR technique with finite element analysis., Methods: Models of clavicle and scapula were constructed using computerized tomography images. Two clavicular bone tunnel reconstruction models were created with the tendon passing through the conoid and trapezoid tunnels. Four models based on the tunnel ratio (TR) method and defined as primary, anatomic, medialized, and lateralized were constructed to evaluate the effect of tunnel placement on loading conditions during tendon graft. All models were loaded by insertion from the trapezius and sternocleidomastoid muscles. The loading on the tendon were evaluated with the finite element analysis., Results: The highest load value measured on the tendon was in the anatomic model (0.789 kPa), and the lowest load value (0.598 kPa) was measured in the lateralized tunnel model. The load value of the primary model was (0.657 kPa), and the medialized model's value was (0.752 kPa)., Conclusions: In two-tunnel CLR technique, tendon loadings are related to tunnel placement. Medialized tunnel placement increases tendon loading. The TR method may be an appropriate option for determining tunnel placement.

We aimed to answer the following three questions in this study: (1) Does tranexamic acid (TA) reduce the amount of bleeding in total knee arthroplasty (TKA)? (2) Does TA reduce requirement for blood transfusion? (3) Is there any difference on reliability and efficacy between topical and intravenous (IV) applications of TA? Patients were allocated into three groups randomly by a software program as topical, IV, and control group. TA was applied as 20 mg/kg to the IV group and as 3 g/100 mL saline to the topical group. The hemoglobin values were recorded preoperatively and postoperatively on the same day and on day 1 and day 2. Removal of the drain postoperatively and length of hospital stay, as well as any complications such as pulmonary embolism or deep venous thrombosis, were also noted. The study comprised 40 patients in the IV group, 42 in the topical group, and 41 in the control group. The drain output values were similar in the IV and topical groups ( p  = 0.161), while those of the control group were significantly higher than both the IV and topical groups ( p  < 0.001 and < 0.001). Transfusion was applied to 19.5% of cases in the control group, 4.8% in the topical group, and 5.1% of the IV group ( χ 2  = 6.522; p  = 0.038). The results of the study showed that 20 mg/kg single-dose IV bolus or 3 g topical TA application reduced blood loss and transfusion requirement without increasing the rate of thromboembolic complications in unilateral primary TKA., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Purpose: Our aim was to evaluate the alterations of subfoveal choroidal thickness (SFCT), photoreceptor layer thickness (PRT), and retinal vessel diameter in the dark and light adaptation., Methods: Twenty-four eyes of 24 healthy volunteers (12 males, 12 females) were included in this cross-sectional and observational study. The SFCT, PRT, retinal arteriole, and venule caliber measurements were performed with spectral domain optical coherence tomography in the dark (0.0 cd/m 2 ) and under light (80 cd/m 2 ) adapted conditions., Results: The mean age of the participants was 30.4 ± 4.4 years (range: 22-42). The SFCT increased statistically significantly in dark adaptation (p < 0.001), then returned to baseline values following light adaptation. The PRT, retinal arteriole, and venule caliber measurements were similar in the dark and light (p > 0.05)., Conclusions: While SFCT increased, PRT, and retinal vessel diameter did not change following transition from light to dark.

Introduction: The aim of this study was to compare the outcome of operated newborns that had an antenatal diagnosis of congenital duodenal obstruction (CDO) with those who had a late diagnosis in the postpartum period., Materials and Methods: The newborns that were operated with the diagnosis of CDO in our department were retrospectively recorded. The patients were grouped according to the time of diagnosis; the ones who had an antenatal diagnosis were assigned to group 1, while the ones that had a diagnosis in the postpartum period were assigned to group 2. The groups were compared in terms of their pregnancy weeks at the time of birth, birth weight, additional congenital anomalies, the type of obstruction, the procedures that are applied, the day of operation, the time for oral feeding tolerance, the duration of hospital stay, and pre- and post-operative complications., Results: Fifteen patients with a diagnosis of CDO were operated on in our department between 2009 and 2014. Eight patients were male and seven patients were female. There were nine patients in group 1 and six patients in group 2. The diagnosis was confirmed in group 1 on the first day of the postpartum period. In the subanalysis, five patients had type 1 CDO and four had type 3 CDO in group 1, while five patients had type 1 CDO and one had type 3 CDO in group 2. There was not any complication in group 1 in the pre-operative period, but two patients had aspiration pneumonia and one had dehydration in group 2 pre-operatively. The mean operation day in the postpartum period was 2.34 (±0.5) days in group 1 and 7.17 (±2.04) in group 2. The time for the patient to tolerate oral feeding in the post-operative period was 11.33 (±1.80) in group 1 and 14.83 (±2.48) in group 2. The duration of hospital stay in group 1 was 20.67 (±9.81) days and 24.66 (±4.50) days in group 2. In the post-operative period, chylous ascites occured in a patient in group 1 and the post-operative period was complicated with ileus in one patient in group 2. No mortalities happened in both groups., Conclusion: The prenatal diagnosis of CDO affects the pre-operative complication rate, the time for the operation in the postpartum period, the duration to start post-operative oral feeding, and the duration of hospital stay, but does not affect the mortality or the morbidity.

Purpose: To evaluate the efficacy and safety of silicone oil barrier sutures in aphakic eyes with iris defects., Methods: Sixteen aphakic and iris-defective eyes of 16 patients who underwent a pars plana vitrectomy procedure with silicone oil tamponade because of retinal detachment were included in this retrospective study. Silicone oil barrier sutures were placed as a grid pattern within the plane of the previous iris after vitrectomy and before silicone oil injection., Results: The mean follow-up time after silicone oil barrier suture operations was 12.0 ± 6.8 months. Silicone oil was present in the anterior chamber in five eyes (31%) at the last visit. These eyes also had hypotony, band keratopathy, and anterior proliferative vitreoretinopathy., Conclusion: In this study, silicone oil barrier sutures were proven to be safe and effective in preventing silicone oil-corneal endothelium touch in aphakic eyes with iris defects, unless hypotony was present because of anterior proliferative vitreoretinopathy.

Purpose: The aim of this study, which is the most extensive series to date on this subject, was to present the results of ten shoulders of seven patients treated with the posterior bone block augmentation method with autograft taken from the iliac wing for a chronic locked posterior shoulder dislocation and to evaluate the results in the light of the relevant literature., Methods: A retrospective examination was made of patients who underwent iliac autograft with glenoid augmentation for chronic posterior shoulder dislocation in our clinic between 2004 and 2011. All of the patients had been initially treated at another centre and referred to our hospital because of continuing or relapsing posterior shoulder dislocation. Assessment was made by physical examination X-ray, computed tomography (CT), and the Constant-Murley score., Results: All the patients were male with a mean age at the time of surgery of 42.4 years (range, 23-53 years), with a mean follow-up period of 40.6 months (range, 24-55 months). On presentation, all the patients had locked posterior shoulder dislocation. Radiological union was evaluated from the CT images taken 24 months postoperatively. The graft was observed to have been incorporated in all cases. The mean postoperative Constant-Murley score was 81.25/100 ± 17.8. In seven of the shoulders, arthropathy findings increased by varying degrees., Conclusions: A functional and stable shoulder can be obtained with glenoid augmentation in patients with chronic locked posterior shoulder dislocation. When other treatment alternatives are considered for a young patient group, this is a safe and applicable treatment method.

Purpose: Idiopathic and diabetic epiretinal membranes (ERMs) differ significantly in terms of macular morphology. Our aim was to compare the photoreceptor outer segment (PROS) length in diabetic and idiopathic ERMs before and after surgery., Methods: Fifty-two eyes of 50 patients who underwent pars plana vitrectomy (PPV) due to idiopathic and diabetic ERMs were included in this retrospective case series. All of the patients underwent ERM and internal limiting membrane (ILM) removal. The patients had been followed up 6 months postoperatively. Spectral-domain optical coherence tomography (OCT) was used to measure the PROS length, overall PR length and central foveal thickness (CFT)., Results: The PROS length, overall PR length, and CFT were statistically significantly lower at all the postoperative visits when compared to preoperative values in both the idiopathic and diabetic ERM groups (P<0.001). The amount of decrease in PROS and overall PR length were similar in both groups after surgery (P>0.05). There was no relation between amount of PROS length change and visual acuity gain in both groups (P>0.05)., Conclusions: Postoperative change of PROS length was similar in idiopathic and diabetic ERMs. PROS length change may not be an indicator of postoperative visual acuity.

Although carotid body tumor (CBT) is a rare neoplasm, it should always be considered in differential diagnosis of lateral neck masses. We shared the 10 years of CBT experience in our clinic and started a discussion on CBT with literature support. A total of 21 patients with CBT diagnosis in Fırat University Hospital, Otorhinolaryngology Clinic, participated in the study. Patients were evaluated based on demographical data and particularities of the tumor. Participant patients were 19 women and 2 men, and their ages were between 21 and 79 (mean age 54.06 ± 12.48). The most frequent reason for the patients to apply to the hospital was painless swelling in the neck (76.2%). Tumor was located in the right side of the neck in 10 patients (47.6%), and in the left side of the neck in 11 (52.4%). Twenty patients (95.2%) had undergone computerized tomography angiography. Surgical treatment was applied to 19 patients (90.5%) and the tumor was totally excised. According to Shamblin classification, 15 of the tumors of these patients were class II (78.9%) and 4 were class III (21.1%). In 1 patient (5.3%), postoperative contusion infection that recovered after medical treatment was observed; in 2 patients (10.5%), n. vagus injury was observed because of tumor's pervasion of n. vagus; and in 1 of these patients vocal cord paralysis was developed and this patient was later taken into thyroplasty surgery. Two patients (10.5%) suffered n. hypoglossus injury, 1 of these recovered within 3 months postoperative and the other developed n. hypoglossus palsy. The size and extension of the tumor should be determined by preoperative imaging for the correct planning of surgical procedure. It should be taken into consideration that despite advanced surgical techniques, the rate of postoperative cranial nerve damage is still high.

Objective: Tularemia is a bacterial zoonosis caused by Francisella tularensis . In this study, we aimed to retrospectively evaluate the documents of patients who had a diagnosis of tularemia in our hospital and discuss this condition with a literature review., Methods: Twenty-five patients who had a diagnosis of tularemia between 2010 and 2013 were included in the study. The patients were assessed with respect to their demographic data; presenting complaints; leukocyte, sedimentation, and C-reactive protein (CRP) values; applied treatment protocols; duration of stay in the hospital; and histopathological examinations., Results: Fifty-seven patients were admitted to the health department of our clinic with a preliminary diagnosis of tularemia, and the diagnosis of tularemia was serologically confirmed for 25 patients. Pharyngeal mucosa cultures, centrifuged serum samples, and abscess and/or pathological samples were collected from all the patients. Thirteen patients were female, and 12 were male. The ages of the patients were between 10 and 75 (average 33.12±15.53) years. The most frequent (100%) symptom among the patients was swelling in the neck. While abscess drainage was performed in 12 patients (48%), the neck mass was excised in 3 patients (12%), and 10 patients (40%) were given only medical treatment. According to the guide of tularemia management by the Ministry of Health, treatment using suitable doses of streptomycin was started for all the patients; in addition, for 8 (32%) patients, doxycycline treatment was added., Conclusion: In recent years, tularemia in our country has been increasing at some specific locations, and those patients are frequently admitted to polyclinics with a complaint of swelling in the neck. In endemic areas in particular, tularemia should also be considered in the differential diagnosis of patients who present with abscess in the neck., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors.

To compare the effect of a single intravitreal injection of triamcinolone acetonide versus three consecutive monthly intravitreal injections of bevacizumab for the treatment of diabetic macular edema (DME). Forty patients treated with a single intravitreal injection of 4 mg triamcinolone acetonide (triamcinolone group) were compared with 40 patients treated with three consecutive monthly intravitreal injections of 1.25 mg bevacizumab (bevacizumab group). The triamcinolone group showed a significant decrease in mean central retinal thickness (CRT) from 472.5 μm ± 120.35 to 374.0 μm ± 152.31 after 3 months (p < 0.001). The bevacizumab group also showed a significant reduction in CRT from 464.5 μm ± 115.3 to 370.0 μm ± 142.31 (p < 0.001). The triamcinolone group displayed an increase in best-corrected visual acuity (BCVA) following a single intravitreal triamcinolone acetonide injection from a mean of +0.70 ± 0.17 logMAR to a mean of +0.54 ± 0.38 logMAR after 3 months (p < 0.05). The bevacizumab group also showed an increase in BCVA following three injections of bevacizumab from a mean of +0.73 ± 0.28 logMAR to a mean of +0.57 ± 0.33 logMAR (p < 0.05). Statistical analysis showed no significant difference between both groups at 3 months. Our study showed that a single intravitreal injection of triamcinolone is as effective as three consecutive monthly intravitreal injections of bevacizumab for treatment of DME with regard to BCVA and CRT measured by optical coherence tomography. Despite the recent popularity of bevacizumab, three injections are no more effective than a single injection of triamcinolone for the treatment of DME.

Purpose: To compare the effects of straight, oblique, and double-plane tunnel scleral intravitreal injection techniques, on short-term intraocular pressure (IOP) changes, occurrence of vitreous reflux (VR), and/or other complications., Methods: A total of 105 patients were included in this prospective study. Patients were assigned randomly to one of the straight injection group (SI; n=35), oblique injection group (OI; n=35), or double-plane tunnel injection group (DPTI; n=35). The occurrence of VR and preinjection and postinjection IOP at 2 and 6 h were compared between groups, and complications were assessed., Results: The mean patient age was 71.1±6.6 years (range 58 to 86 years; 50 women and 55 men). In the SI, OI, and DPTI groups, VR occurred in 18 (51.4%), 12 (34.3%), and 6 patients (17.1%), respectively. Mean preinjection IOPs were 15.4, 14.8, and 15.6 mmHg, and changed to 17.3, 18.0, and 20.0 mmHg at the second hour, and 15.7, 16.2, and 16.1 mmHg at the sixth hour, respectively. No significant differences among the groups were found in terms of complications such as transient mild uveitis or subconjunctival hemorrhage., Conclusion: Double-plane tunnel injection prevents VR from the injection site and has no additional complications than other techniques.

Unlabelled: PUPOSE: The purpose of this study was to compare the biomechanical properties of the cornea and intraocular pressure (IOP) between patients with systemic lupus erythematosis (SLE) and age-matched controls., Patients and Methods: In this prospective study, 30 healthy individuals (control group) and 30 patients with SLE (study group) underwent Reichert ocular response analyzer (ORA) measurements. In the right eye of each participant, the corneal hysteresis (CH), corneal resistance factor (CRF), and Goldman-related IOP (IOPg) were recorded using the ORA., Results: Mean CH, CRF, IOPg were significantly different between groups. Mean CH was 10.2 ± 0.6 mm Hg in the study group and 11.3 ± 1.3 in the control group (P=0.02); mean CRF was 9.7 ± 1.1 mm Hg and 11.9 ± 1.5 mm Hg, respectively (P=0.001). Mean IOP(g) was 13.9 ± 2.9 mm Hg in the study group and 16.9 ± 2.6 mm Hg in the control group (P=0.001)., Conclusion: The biomechanical properties of the cornea are altered in patients with SLE compared with normal controls. These findings should be taken into account when measuring IOP values in patients with SLE as IOP readings may be underestimated in SLE eyes.

Objective: A standard top-loading lumbar pedicle screw-rod system is compared with a pedicle screw-plate system with smaller-diameter screws, more medial entry, and lower profile to assess the relative stability, strength, and resistance to fatigue of the 2 systems., Methods: Seven human cadaveric specimens were studied with each surgical construct. Nondestructive, nonconstraining pure moments were applied to specimens to induce flexion, extension, lateral bending, and axial rotation while recording L5-S1 motion optoelectronically. After initial tests, specimens were fatigued for 10,000 cycles and retested to assess early postoperative loosening. Specimens were then loaded to failure in hyperextension., Results: The standard screw-rod construct reduced range of motion to a mean of 20% of normal, whereas the screw-plate construct reduced range of motion to 13% of normal. Differences between systems were not significant in any loading mode (P > 0.06). The 14% loosening of the screw-rod system with fatigue was not significantly different from the 10% loosening observed with the screw-plate system (P > 0.15). Mean failure loads of 30 Nm for screw-rod and 37 Nm for screw-plate were also not significantly different (P = 0.38)., Conclusion: Posterior fixation at L5-S1 using the low-profile screw-plate system offers stability, resistance to fatigue, and resistance to failure equivalent to fixation using a standard cantilevered pedicle screw-rod system.

Object: An experiment was performed to study the limits of the ability of screws designed to center themselves in the pedicle during insertion, and to study whether straight-ahead versus inward screw insertion trajectories differ in their resistance to pullout., Methods: Forty-nine human cadaveric lumbar vertebrae were studied. Pedicle screws were inserted in trajectories starting 0 degrees, 10 degrees, 20 degrees, or 30 degrees from the optimal trajectory, either medially or laterally misdirected. The surgeon then inserted the screw with forward thrust but without resisting the screw's tendency to reorient its own trajectory during insertion. On the opposite pedicle, a control screw was inserted with the more standard inward-angled anatomical trajectory and insertion point. Cortical wall violation during insertion was recorded. Screws were then pulled out at a constant displacement rate while ultimate strength was recorded., Results: Lateral misdirection as small as 10 degrees was likely to lead to cortical wall violation (3 of 7 violations). Conversely, medial misdirection usually resulted in safe screw insertion (1 of 21 violations for 10 degrees, 20 degrees, or 30 degrees medial misdirection). The resistance to pullout of screws inserted in a straight-ahead trajectory did not differ significantly from that of screws inserted along an inward trajectory (p = 0.68)., Conclusions: Self-tapping, self-drilling pedicle screws can redirect themselves to a much greater extent during medial than during lateral misdirection. The cortical wall is more likely to be violated laterally than medially. The strength of straight-ahead and inward trajectories was equivalent.

Study Design: Biomechanical load-to-failure findings correlated with anatomic dissection measurements and intact (prefailure) 3-Tesla (3-T) magnetic resonance images (MRI)., Objective: To better understand why the same distractive force to the head can result in occipitoatlantal dislocation (OAD) in some individuals and atlantoaxial dislocation (AAD) in others., Summary of Background Data: Distraction injuries to the cranio-vertebral junction have been studied biomechanically but have not been studied relative to ligamentous anatomic variations. We theorized that morphologic variations in the ligaments should influence the injury pattern during axial distraction., Methods: After obtaining 3-T MRI scans, 10 occiput-C2 specimens were loaded to failure under axial tension. Direct anatomic measurements were also obtained from the distracted and injured specimens., Results: AAD was observed in 7 specimens (mean force +/- standard deviation 1229 +/- 181 N) at a significantly higher magnitude than OAD, which was observed in 3 specimens (823 +/- 127 N; P = 0.009, nonpaired t test). In OAD specimens, the superior cruciate ligament (SCL), which was smaller than the inferior cruciate ligament (ICL), failed. The apical ligament was unidentifiable in these 3 specimens. In 5 of the 7 AAD specimens, the ICL ruptured and was smaller than the SCL. In the remaining 2 specimens, both SCL and ICL ruptured. The apical ligament, which ruptured, was identifiable in all 7 specimens., Conclusion: Axial distraction across the cranio-vertebral junction can produce either OAD or AAD. The SCL and ICL dimensions, alar ligament orientations, and apical ligament presence may affect the injury site. Visualization with 3-T MRI allows better understanding of the injury mechanism and location, which is important clinically in selecting single- or multilevel fixation.

Object: The stability provided by 3 occipitoatlantal fixation techniques (occiput [Oc]-C1 transarticular screws, occipital keel screws rigidly interconnected with C-1 lateral mass screws, and suboccipital/sublaminar wired contoured rod) were compared., Methods: Seven human cadaveric specimens received transarticular screws and 7 received occipital keel-C1 lateral mass screws. All specimens later underwent contoured rod fixation. All conditions were studied with and without placement of a structural graft wired between the skull base and C-1 lamina. Specimens were loaded quasistatically using pure moments to induce flexion, extension, lateral bending, and axial rotation while recording segmental motion optoelectronically. Flexibility was measured immediately postoperatively and after 10,000 cycles of fatigue., Results: Application of Oc-C1 transarticular screws, with a wired graft, reduced the mean range of motion (ROM) to 3% of normal. Occipital keel-C1 lateral mass screws (also with graft) offered less stability than transarticular screws during extension and lateral bending (p < 0.02), reducing ROM to 17% of normal. The wired contoured rod reduced motion to 31% of normal, providing significantly less stability than either screw fixation technique. Fatigue increased motion in constructs fitted with transarticular screws, keel screws/lateral mass screw constructs, and contoured wired rods, by means of 19, 5, and 26%, respectively. In all constructs, adding a structural graft significantly improved stability, but the extent depended on the loading direction., Conclusions: Assuming the presence of mild C1-2 instability, Oc-C1 transarticular screws and occipital keel-C1 lateral mass screws are approximately equivalent in performance for occipitoatlantal stabilization in promoting fusion. A posteriorly wired contoured rod is less likely to provide a good fusion environment because of less stabilizing potential and a greater likelihood of loosening with fatigue.

Object: An in vitro flexibility experiment was performed to compare the biomechanical stability of asymmetrical lumbar pedicle screw fixation (longer hardware attached ipsilaterally to a 1-sided lesion), short and long fixation, and fixation with and without interconnection to the involved vertebra., Methods: Seven human cadaveric specimens (T12-S1) were studied intact; after simulated unilateral lesions were created at L2-3 and L3-4, the segments were stabilized by 1) L2-4 unilateral fixation (L-3 excluded), 2) L2-4 bilateral fixation (L-3 included contralaterally), 3) L2-5 unilateral fixation (L-3 excluded), 4) L2-5 fixation ipsilateral (L-3 excluded) and L2-4 fixation contralateral (L-3 included), 5) L2-5 bilateral fixation (L-3 included contralaterally), and 6) L2-5 bilateral fixation (L-3 excluded). The testing order varied among specimens. Angular range of motion (ROM) and lax zone were recorded optically while loading to 6.0 Nm was created with nonconstraining pure moments., Results: Unilateral short fixation provided significantly worse stabilization than any other construct tested in all loading modes (p < 0.05, repeated-measures analysis of variance). There was a mean 56% reduction in ROM across the lesion after adding 1 additional level rostrally and caudally. Asymmetrical long/short stabilization provided similar stability to symmetrical long stabilization. Minimal additional stability was gained by including L-3 in the long bilateral fixation construct., Conclusions: Unilateral fixation is inadequate for stabilizing a 2-level unilateral lesion. Bilateral fixation, whether symmetrical or asymmetrical, provides good stabilization for this injury. It is not important for stability to include the level of the lesion within the long construct contralaterally.

Background Context: Extrapedicular screws are placed more laterally than intrapedicular screws and pass through the transverse process or rib head before entering the vertebral body. These screws are sometimes placed to salvage failed pedicle screws, but the change in pullout resistance of extrapedicular screws after salvage has not been quantified., Purpose: To quantify the pullout resistance of thoracic extrapedicular screws compared with intrapedicular screws and the pullout resistance of newly inserted screws compared with extrapedicular screws used as salvage for failed intrapedicular screws., Study Design: In vitro paired comparison of screw pullout resistance in isolated thoracic vertebrae., Methods: Tapered monoaxial pedicle screws were inserted in the left or right pedicle of 11 human cadaveric thoracic vertebrae. An extrapedicular screw was inserted on the contralateral side. Both screws were pulled out axially at 0.5 mm/s using a servohydraulic test frame while applied load was recorded. Then a fresh extrapedicular screw was inserted as a salvage screw on the intrapedicular screw side and pulled out., Results: In uncompromised vertebrae, the pullout strength of extrapedicular screws was 80+/-32% of that of intrapedicular screws (p=.073, repeated-measures one-way analysis of variance/Tukey). Salvage screws restored pullout strength to 65+/-30% of that of intrapedicular screws (p=.003)., Conclusions: Extrapedicular screws provided comparable but slightly lower pullout resistance to intrapedicular screws in uncompromised vertebrae. They are therefore a feasible salvage technique when a compromised pedicle precludes reinsertion of an intrapedicular screw, but the salvage screw is significantly weaker than the original screw.

Background Context: The technique of occipitocervical fusion using a threaded contoured rod attached with sublaminar wires to the occiput and upper cervical vertebrae is widely used throughout the world and has been clinically proven to provide effective fixation of the destabilized spine. However, this system has some disadvantages in maintaining stability, especially at C1-C2 because of the large amount of axial rotation at this level. In some clinical situations such as fracture of the C1 lamina, C1 laminectomy, and excessively lordotic curvature, it is not always possible to wire C1 directly into the construct. In such cases, combination of other stabilization methods that include C1 indirectly can be used to achieve a reliable posterior internal fixation., Purpose: Primarily, to evaluate whether a contoured rod construct in which C1 is indirectly included using C1-C2 transarticular screws is biomechanically equivalent to a standard, fully wired contoured rod construct. Secondarily, to evaluate the biomechanical benefit of adding C1-C2 transarticular screws to a fully wired contoured rod construct., Study Design: Repeated-measures nondestructive in vitro biomechanical testing of destabilized cadaveric human occipitocervical spine specimens., Methods: Six human cadaveric specimens from the occiput to C3 were studied. Angular and linear displacement data were recorded while nonconstraining nondestructive loads were applied. Three methods of fixation were tested: contoured rod incorporating C1 with and without transarticular screws and contoured rod with transarticular screws without incorporating C1., Results: All three constructs reduced motion to well within normal range. In contoured rod constructs with C1 wired, addition of transarticular screws slightly but significantly improved stability. In constructs with transarticular screws, incorporation of C1 into the contoured rod wiring did not improve stability significantly., Conclusions: Adding C1-C2 transarticular screws to a wired contoured rod construct where C1 is included only slightly improves stability. As the absolute reduction in motion from adding transarticular screws is small (<1 degree), it is doubtful whether any enhanced fusion from this additional procedure outweighs the surgical risks. However, transarticular screws provide an effective alternate method to fixate C1 when the posterior arch of C1 is absent or has been fractured.

We performed a prospective randomized study to compare maternal and fetal outcomes in pregnancies with prelabour rupture of the membranes (PROM) at term with early induction of labour or expectant management, 126 women with singleton pregnancy, cephalic presentation and gestational duration > or = 37 weeks, were randomized either to immediate induction of labour with oxytocin (Group 1) (n=52), or conservative management (Group 2) (n=74). Women who constituted Group 2 were divided into 2 groups. The first group (Group 2A) (n=25) included women in whom spontaneous labour did not begin after a waiting period of 24 hours, in which case labour was induced with oxytocin i.e. expectant management. The second group consisted of women (Group 2B) (n=49) in whom labour began spontaneously within 24 hours. The base Caesarean section rate was significantly higher in Group 2 (28.4%) (p<0.05). The rates of Caesarean section in the Groups 1-2A-2B were 19.2%, 60%, and 12.2%, respectively for nulliparous and parous women together. The rate of fetal distress was significantly higher in Group 2 (p<0.05). For determining maternal outcomes, the other parameters such as clinical chorioamnionitis, fever before or during labour, receiving antibiotics before or during labour, postpartum fever, analgesia, anaesthesia did not differ in Groups 1 and 2. Women in Group 1 went into active labour sooner, had fewer digital vaginal examinations, had a shorter interval between membrane rupture and delivery, and spent less time in the hospital before delivery than those in Group 2 (p<0.05). Babies in Group 2 were more likely to receive antibiotics, and more likely to stay in an intensive care nursery for more than 24 hours, and more likely to receive ventilation after initial resuscitation than those babies in Group 1. For developing apnoea and hypotonia, there was no significant difference between Groups 1 and 2. However, for babies in Group 2A there was a significant difference. We conclude that immediate induction of labour with oxytocin does not increase the risk of Caesarean section, compared with a practice of expectant management. Women at term with prelabour rupture of the membranes should therefore be reassured that immediate induction with oxytocin currently appears to be the best policy with respect to maternal and neonatal morbidity.

A case of multiple hydatid cysts, of embolic origin from a cyst of the left atrium, is discussed. The patient was first admitted to hospital with an intracerebral haemorrhage and was treated conservatively. There was not any cystic or mass lesion other than intracerebral haemorrhage on his computed tomography (CT) scans. In the follow-up period, the CT scans after 14 months showed multiple hydatid cysts, with the largest about 6 cm in diameter, which suggested a growth rate of 5 cm per year.

Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer. These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme. A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations. The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment. Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation. Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface. These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea. The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis. Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15. These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.

Triosephosphate isomerase (TPI) provides an excellent model for terminal marking and protein degradation. Mammalian TPI is terminally modified by deamidation at Asn71-Gly, resulting in unfolding, dissociation, and proteolysis. In contrast, chicken TPI, which contains a lysine at position 71, is terminally modified by the oxidation of Cys126. Thus, both deamidation and oxidation initiate degradation of TPI from different species. To explore the terminal marking in other avians, we have purified the turkey TPI to homogeneity and determined its characteristics. Although the molecular properties of the turkey and chicken TPI were very similar, their tolerances to temperature, oxidants, and alkaline pH were very different. For example, chicken TPI was inactivated 80% in either 10 mM oxidized glutathione or H2O2, whereas 120 mM GSSG had no effect on turkey TPI, and > 120 mM H2O2 was needed for 80% inactivation. Under alkaline conditions that cause rapid deamidation of the mammalian TPI, neither avian enzyme deamidated. Chicken TPI, however, aggregated. Aggregation was reversed by 2-mercaptoethanol. Under prolonged exposure to milder conditions the turkey enzyme was completely inactivated and deamidated at Asn15-Gly. Thus, there are marked differences in the susceptibility of these two avian enzymes to oxidation and deamidation, and their terminal marking mechanisms appear to be different.

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.

Covalent modification of Glu165 in the catalytic center of triose-phosphate isomerase with the substrate analogue 3-chloroacetol phosphate traps the complex in two conformations. The two resulting 31P NMR resonances at 6.9 and 5.7 ppm appear to reflect conformations in which the hinged lid (residues 167-176) is in the open and closed positions. The conformation represented by the 5.7-ppm resonance is more stable, and unfolding and refolding in guanidine converts all of the molecules to the 5.7-ppm conformation. The complete conformational transition from 6.9 to 5.7 ppm also takes place as a function of time and temperature. Under these conditions the native enzyme retains more than 80% of the catalytic activity, indicating that this conversion is not due to thermal denaturation of the enzyme. Circular dichroic and fluorescence spectroscopy indicate that the 3-chloroacetol phosphate-modified enzyme does not undergo major structural changes. From the temperature dependence of this transition, an energy barrier of 144 kJ/mol (34.4 kcal/mol) was calculated for this conversion.

Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.

Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.

The accumulation of modified proteins in aging is well documented in many aging models. For example, the deamidated isoforms of triosephosphate isomerase accumulate in: (a) old erythrocytes, (b) fibroblasts from old donors, (c) fibroblasts aged in vitro, (d) premature-aging syndromes and (e) old cells in the eye lens. However, a fundamental remaining question is: 'Do such modified proteins interfere with cellular function?' It has been difficult to assess this question at the molecular level using whole-organism models and equally frustrating to evaluate the physiological significance of such changes using classical cellular models. Tissue equivalent systems (TES) provide an opportunity for examining the molecular basis and physiological consequences of modified proteins during aging. TES are composed of differentiating and proliferating heterogeneous cell types with symbiotic cell-cell and cell-matrix interactions. They closely resemble, both morphologically and functionally, the tissues from which they were derived. Aging studies utilizing TES can provide information on modifications of protein structures, isozyme patterns, enzymes of the cellular environmental protection system and metabolic parameters which may regulate protein synthesis and degradation.

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.

The electrophoretic isoforms of mammalian triosephosphate isomerase (TPI; EC 5.3.1.1) are due to deamidation at two Asn-Gly sites (Asn15 and Asn71). Deamidation of these two asparagines in the subunit-subunit interface of the isologous dimer appears to destabilize the dimer and initiate degradation of the protein. Chicken TPI contains a lysine substitution for Asn71, thus precluding this deamidation site. Nevertheless, the chicken enzyme exhibits three electrophoretic isoforms. This multiplicity is not the result of deamidation of the remaining Asn15 site, but due to a specific site which is highly susceptible to oxidation. The three isoforms of chicken TPI can be reduced to a single form in the presence of high concentrations of reducing agents (e.g., greater than 15 mM dithiothreitol or greater than 50 mM 2-mercaptoethanol) and are also generated when oxidizing agents, such as oxidized glutathione, are present. The oxidized isoforms exhibit lowered catalytic activity and are more susceptible to denaturation and proteolytic degradation than the native enzyme. Structural analysis of the isoforms by chemical cleavage at the cysteine peptide bonds with 2-nitro-5-thiocyanobenzoic acid and subsequently at the methionines with CNBr followed by peptide sequencing reveals that Cys126 is the site of the modification. Since the oxidized isoforms of chicken TPI accumulate in vivo during aging analogous to the deamidated isoforms from mammals, it appears that TPI is the first example of a protein which has evolved two specific types of weak links which may initiate turnover of the protein.

Triosephosphate isomerase (TPI) from mammals undergoes two specific deamidations (Asn-15 and Asn-71) which destabilize the isologous dimer and lead to the degradation of the protein. In aging cells and tissues, the deamidated isoforms accumulate apparently due to age-related changes in protein turnover. Chicken TPI lacks one of these sites (i.e., Asn 71----Lys), but also exhibits unstable isoforms. These isoforms are the result of the specific oxidations which occur both in vitro and in vivo. Electrophoretic analyses of various tissues from chicken show that the most oxidized isoform, which is present in adult tissues, is only present in small quantities in tissues of the newborn chick. Moreover, embryonic tissues contain almost exclusively the fully reduced form of TPI. Thus, it appears that oxidation rather than deamidation constitutes the first step in the degradation of avian TPI. TPI may be the first example of a protein which has evolved two different types of modifications (deamidation and oxidation) which trigger its degradation. The accumulation of both deamidated and oxidized isoforms in different species may provide clues to the underlying basis for the accumulation of modified proteins in aging.

Amino acid compositions of skin samples from young and old subjects and from age-matched donors with dry skin syndrome (xerosis) were examined. The amino acid contents of the free amino acid (FAA) fraction, soluble hydrolysate (SH) fraction, and whole cell hydrolysate (WCH) were determined. The greatest differences were observed between the FAA compositions of the young and old normal subjects. Xerosis did not appear to affect the amino acid compositions of samples from young subjects as much as old subjects. Overall, the effect of aging on the amino acid contents was more pronounced than the effect of xerosis. The amino acid composition of the FAA showed a high degree of similarity to filaggrin, whereas the WCH showed a similarity to keratin.

A new method for the isolation of homogeneous triosephosphate isomerase (TPI, EC 5.3.1.1) has been developed. The method utilizes high-performance liquid chromatography on DEAE 5PW and Hydrophase-polyethyleneimine columns, which results in the rapid isolation and essentially quantitative recovery of the enzyme. The procedure is superior to previous methods with respect to specificity, recovery, and time. In addition, this rapid process minimizes the potential for postsynthetic modifications of the protein. Milligram quantities of TPI can be isolated from 100 g of tissue.

The effects of pH, temperature, buffer ion, ionic strength, protein concentration, and substrate on the rates of specific, spontaneous deamidations of Asn-15 and Asn-71 of human triosephosphate isomerase were examined. Elevated temperature and pH facilitate the deamidations, and the deamidation rate is dependent on the specific buffer ions indicating a general base catalysis mechanism. The presence of substrate also enhances the rates of deamidation. The effect of substrate may be related to conformational changes in the catalytic center which are known to cause changes in the subunit-subunit contact sites where Asn-15 and Asn-71 are located. The enhanced deamidation in the presence of substrate may, in part, account for the more rapid rate of deamidation observed in vivo.