Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis.

Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.

Cellular and Molecular Mechanisms of Pathogenesis Underlying Inherited Retinal Dystrophies.

Inherited retinal dystrophies (IRDs) are congenital retinal degenerative diseases that have various inheritance patterns, including dominant, recessive, X-linked, and mitochondrial. These diseases are most often the result of defects in rod and/or cone photoreceptor and retinal pigment epithelium function, development, or both. The genes associated with these diseases, when mutated, produce altered protein products that have downstream effects in pathways critical to vision, including phototransduction, the visual cycle, photoreceptor development, cellular respiration, and retinal homeostasis. The aim of this manuscript is to provide a comprehensive review of the underlying molecular mechanisms of pathogenesis of IRDs by delving into many of the genes associated with IRD development, their protein products, and the pathways interrupted by genetic mutation.

LncRNA MALAT1 Modulates TGF-ß1-Induced EMT in Keratinocyte.

One of the major complications in diabetes is impaired wound healing. Unfortunately, effective therapies are currently lacking. Epithelial to mesenchymal transition (EMT) is a critical process involved in cutaneous wound healing. In response to injury, EMT is required to activate and mobilize stationary keratinocytes in the skin toward the wound bed, which allows for re-epithelialization. This process is stalled in diabetic wounds. In this study, we investigate the role of long non-coding RNA (lncRNA), MALAT1, in transforming growth factor beta 1(TGF-ß1)-induced EMT of human keratinocyte (HaCaT) cells. Initially, we detected MALAT1 and TGF-ß1 expression in non-diabetic and diabetic wounds and found that these expression are significantly up-regulated in diabetic wounds. Then, HaCaT cells were cultured and exposed to TGF-ß1. The EMT of HaCaT cells were confirmed by the increased expression of CDH2, KRT10, and ACTA2, in addition to the down-regulation of CDH1. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). MALAT1 silencing attenuates TGFß1-induced EMT. Mechanistically, MALAT1 is involved in TGF-ß1 mediated EMT through significantly induced ZEB1 expression, a critical transcription factor for EMT. In summary, lncRNA MALAT1 is involved in TGFß1-induced EMT of human HaCaT cells and provides new understanding for the pathogenesis of diabetic wounds.

Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy.

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.

Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic or random free radical reaction?

Duality of iron as an essential cofactor of many enzymatic metabolic processes and as a catalyst of poorly controlled redox-cycling reactions defines its possible biological beneficial and hazardous role in the body. In this review, we discuss these two "faces" of iron in a newly conceptualized program of regulated cell death, ferroptosis. Ferroptosis is a genetically programmed iron-dependent form of regulated cell death driven by enhanced lipid peroxidation and insufficient capacity of thiol-dependent mechanisms (glutathione peroxidase 4, GPX4) to eliminate hydroperoxy-lipids. We present arguments favoring the enzymatic mechanisms of ferroptotically engaged non-heme iron of 15-lipoxygenases (15-LOX) in complexes with phosphatidylethanolamine binding protein 1 (PEBP1) as a catalyst of highly selective and specific oxidation reactions of arachidonoyl- (AA) and adrenoyl-phosphatidylethanolamines (PE). We discuss possible role of iron chaperons as control mechanisms for guided iron delivery directly to their "protein clients" thus limiting non-enzymatic redox-cycling reactions. We also consider opportunities of loosely-bound iron to contribute to the production of pro-ferroptotic lipid oxidation products. Finally, we propose a two-stage iron-dependent mechanism for iron in ferroptosis by combining its catalytic role in the 15-LOX-driven production of 15-hydroperoxy-AA-PE (HOO-AA-PE) as well as possible involvement of loosely-bound iron in oxidative cleavage of HOO-AA-PE to oxidatively truncated electrophiles capable of attacking nucleophilic targets in yet to be identified proteins leading to cell demise.

Comparison of Descemet stripping under continuous air flow, manual air injection and balanced salt solution for DMEK: a pilot study.

BACKGROUND: Descemet's membrane endothelial keratoplasty (DMEK) involves removal of the recipient's Descemet membrane (DM) prior to transplanting the donor's DM. When using balanced salt solution (BSS) or ophthalmic viscosurgical devices (OVDs), visualization of the host's DM during its stripping may be inadequate and may result in Descemet remnants and could lead to sub-optimal surgical results. Previous articles described excellent visualization when utilizing air injection but this requires repeated air injection into the anterior chamber (AC). We present a pilot study that compares different techniques under which DM stripping can be performed: with continuous automated air infusion, with manual air infusion, and with BSS. METHODS: We retrospectively compared video footage of DM stripping with BSS, with continuous air and with manual injection of air into the AC to determine DM stripping duration and the number of times the surgeon had to insert and retrieve a surgical instrument from the AC. RESULTS: Thirty videos of 10 consecutive cases of the three DM stripping techniques were evaluated. DM stripping duration was 3.26 (±1.32), 3.92 (±1.2) and 12.9 (±3.98) minutes for BSS, continuous air flow, and manual air injection, respectively. Frequency of instrument retrieval (FIR) was 3.6 (±1.71), 1.5 (±0.71) and 15.1 (±3.28) for BSS, continuous air flow, and manual air injection, respectively. Continuous air flow and BSS were both statistically different than manual air injection into the AC (p < 0.05), but did not differ from one another statistically. CONCLUSION: DM stripping during posterior lamellar surgery is imperative for favorable post-operative results and prevention of complications. Performing this step under air in the AC contributes to better visualization and an efficient surgery.

[Galleria mellonella larva model in evaluating the effects of biofilm in Candida albicans]. / Candida albicans biyofilm etkilerinin degerlendirilmesinde Galleria mellonella larvasi modeli.

Biofilm-related infections are chronic infections that cause serious increase in morbidity and mortality as well as significant economic loss. Galleria mellonella larva is shown as a reliable animal model for in vivo toxicology and pathogenicity tests due to its large size, ease of practice, ability to survive at 15-37°C and its similarity to mammals' natural immune system. The aim of this study was to evaluate the effects biofilm activity of Candida albicans in a G.mellonella larva model. Two C.albicans strains isolated as a disease agent were used for the model, where one was positive (BP), and the other one was negative (BN) for biofilm production. Eighty healthy G.mellonella larvae, all in the last larval stage and 2-2.5 cm long, were divided into 4 groups of equal size. Group 1 was set as the control group. Group 2 was injected with sterile phosphate buffer (PBS) group. Group 3 was injected with BP C.albicans strain and group 4 with BN C.albicans strain. A 5 µL volume of C.albicans prepared at 5 × 105 cfu/ml concentration with PBS was injected into the last left rear-legs of the larvae. The larvae were kept in sterile petri dishes at 37°C. They were observed for a total of 96 hours, for 4 hours in the first 24 hours, then in 12 hours intervals. Melanization, survival, total hemocyte count and fungal burden were evaluated as infection indicators. Melanization and death were not observed throughout the study period in group 1. One larva died in group 2. Small melanization spots (dark spots) and subsequent progressive melanization were observed from 3rd hour in the larvae infected with C.albicans. When compared with the BN C.albicans infected group, survival rate was 20% for BP C.albicans infected larvae at the end of 24 hours. Total hemocyte count was very low in the infected groups compared to groups 1 and 2, also significantly lower in group 3 than in group 4. In quantitative cultures, growth of C.albicans was detected in groups 3 and 4 while not in groups 1 and 2. Fungal load was significantly higher in BP C.albicans infected group than BN C.albicans infected group. In this study, G.mellonella larvae were used as live hosts to demonstrate the effects of biofilm activity of C.albicans. Our results suggest that larval models can be used to investigate the effects of fungal infections and biofilm like virulence factors on host cells, and invertebrate animal models can be widely used and can bridge between in vitro studies and mammalian models.

Validation of the EORTC QLQ-BIL21 questionnaire for measuring quality of life in patients with cholangiocarcinoma and cancer of the gallbladder.

BACKGROUND: There is no specific quality of life (QoL) measurement tool to quantify QoL in patients with biliary tract cancer. Quality of life measurement is an increasingly crucial trial end point and is now being incorporated into clinical practice. METHODS: This International Multicentre Phase IV Validation Study assessed the QLQ-BIL21 module in 172 patients with cholangiocarcinoma and 91 patients with cancer of the gallbladder. Patients completed the questionnaire at baseline pretherapy and subsequently at 2 months. Following this, the psychometric properties of reliability, validity, scale structure and responsiveness to change were analysed. RESULTS: Analysis of the QLQ-BIL21 scales showed appropriate reliability with Cronbach's α-coefficients >0.70 for all scales overall. Intraclass correlations exceeded 0.80 for all scales. Convergent validity >0.40 was demonstrated for all items within scales, and discriminant validity was confirmed with values <0.70 for all scales compared with each other. Scale scores changed in accordance with Karnofsky performance status and in response to clinical change. CONCLUSIONS: The QLQ-BIL21 is a valid tool for the assessment of QoL in patients with cholangiocarcinoma and cancer of the gallbladder.

Global motions exhibited by proteins in micro- to milliseconds simulations concur with anisotropic network model predictions.

The Anton supercomputing technology recently developed for efficient molecular dynamics simulations permits us to examine micro- to milli-second events at full atomic resolution for proteins in explicit water and lipid bilayer. It also permits us to investigate to what extent the collective motions predicted by network models (that have found broad use in molecular biophysics) agree with those exhibited by full-atomic long simulations. The present study focuses on Anton trajectories generated for two systems: the bovine pancreatic trypsin inhibitor, and an archaeal aspartate transporter, GltPh. The former, a thoroughly studied system, helps benchmark the method of comparative analysis, and the latter provides new insights into the mechanism of function of glutamate transporters. The principal modes of motion derived from both simulations closely overlap with those predicted for each system by the anisotropic network model (ANM). Notably, the ANM modes define the collective mechanisms, or the pathways on conformational energy landscape, that underlie the passage between the crystal structure and substates visited in simulations. In particular, the lowest frequency ANM modes facilitate the conversion between the most probable substates, lending support to the view that easy access to functional substates is a robust determinant of evolutionarily selected native contact topology.

[Evaluation of the Relationship Between Microenvironment and Biofilm and Coagulase Responses of Staphylococcus aureus Strains Under the Effect of Gentamicin]. / Gentamisin Etkisi Altindaki Staphylococcus aureus Suslarinin Biyofilm ve Koagülaz Yanitlari ile Mikroçevre Iliskisinin Degerlendirilmesi.

The frequency of infections caused by Staphylococcus aureus strains and the rate of antibiotic resistance among these strains are gradually increasing. Accordingly, serious problems emerge in the treatment of community or hospital acquired S.aureus infections. This study was aimed to determine the role of MIC and sub-MIC concentrations of gentamicin on biofilm and coagulase forming effects of S.aureus in in vitro test systems and cell cultures. A standard S.aureus ATCC 25923 strain and two clinical S.aureus strains isolated from blood cultures (C1 and C2) were included in the study. Gentamicin MIC values of the strains were determined with microdilution method at the cation-adjusted Mueller Hinton broth according to CLSI standards. For each strain, MIC, 50% MIC and 25% MIC values of gentamicin were determined separately. At the determined MIC values, biofilm formations of strains were determined with crystal violet method spectrophotometrically. Also, coagulase activities of the strains were evaluated in glass tubes. Human origin epithelial cell cultures namely HEp-2 cell lines, were infected with the standard and clinical S.aureus strains (Multiplicity of infection: 50/1) and left for incubation for two hours. After all, MIC, 50% MIC and 25% MIC values of gentamicin, were added to infected cell lines and incubated for 18 hours. Cells were blown up with distilled water and then bacteria were collected. Biofilm formation and coagulase production of these bacteria were evaluated. When S.aureus ATCC 25923 strain and C1 strains' biofilm formation was evaluated before (in vitro) and after incubation in cell culture, no difference was observed. However in C2 strain, under the effect of MIC level gentamicin, biofilm formation was occurred after interaction with the cell. In the same way, when coagulase responses were evaluated, after interaction with the cell, coagulase production of C2 strain was inhibited. These results indicated that, phenotypic characteristics such as biofilm formation and coagulase production might change during the process of bacterial adaptation to microenvironment. Further advanced experimental modelling designed with different combinations of antibiotics and different cell lines may provide data about the causes and timing of these phenotypic changes and shed light on the development of new treatment policies.

[Experimental oral candidiasis in healthy and immunocompromised BALB/c mice]. / Saglikli ve Immün Sistemi Baskilanmis BALB/c Farelerde Deneysel Oral Kandidiyaz.

Oral candidiasis which is the most common type of Candida infections affecting humans, is most frequently caused by C.albicans. Immune response of the host, as well as a variety of virulence factors of the causative agent, play important roles in the development of Candida infections. The colonization rate of Candida in the oral cavity of healthy individuals, is between 25-30%, however, this rate is reported to be increased in immunosuppressive subjects. In our study, we established an oral candidiasis model with C.albicans in healthy and experimentally immunocompromised mice and aimed to compare Candida colonization rates and histopathological changes occurred in the tongue and esophagus tissues of the animal groups. A total of 21 BALB/c mice were grouped as control (Group 1; n= 7), healthy (Group 2; n= 7) and immunocompromised (Group 3; n= 7) groups. Immunosuppression in mice was performed by subcutaneous injection of prednisolone. For experimental oral candidiasis, cotton swab impregnated with C.albicans strains which did not have acid proteinase and phospholipase enzyme activity, no biofilm production, and sensitive to fluconazole and amphotericin B, were used. In the control group, physiological saline solution was used instead of C.albicans strain. In the forth day of experimental oral candidiasis model swab samples taken from the dorsal tongue surface of mice were evaluated by quantitative cultivation method. No yeast colonies were detected in Group 1 while more significant number of yeast colonies were observed in Group 3 compared to Group 2 (p= 0.002). Tongue and esophagus tissues of mice were stained with hematoxylin-eosin and periodic acid schiff staining and evaluated in terms of inflammatory response, abscess formation, vascular congestion, vasodilation and for the presence of yeast and hyphae. When the inflammation in esophagus was considered, statistically significant difference was determined between group 1 and group 3 (p= 0.023), however, no difference was detected between group 2 and 3 (p= 0.107). The level of inflammation in tongue tissue exhibited no difference between groups 2 and 3 (p= 0.317) while the difference was significant when these groups were compared to the control group (p= 0.00, p= 0.002, respectively). Similarly, the level of congestion in tongue tissue exhibited no difference between groups 2 and 3, however, the difference was significant when compared to the control group. To enlighten the relation between host immune status and oral candidiasis caused by C. albicans, further larger-scale studies also concerning the various virulence factors of the infectious agent, should be conducted by the use of experimental animal models which may successfully guide us in this regard.

Half top hat wound configuration for penetrating keratoplasty: 1-year results.

AIMS: To compare the 1-year outcomes after half-top-hat (HTH) penetrating keratoplasty (PK) versus top-hat (TH) PK and regular PK. METHODS: We reviewed the clinical notes of 87 consecutive patients who had undergone either HTH PK (23 eyes), TH PK (36 eyes) or regular PK (35 eyes) at Toronto Western Hospital between 2002 and 2007. We evaluated best-corrected visual acuity (BCVA), topographic and refractive results, high-order ocular aberrations, endothelial cell counts and complication rates. RESULTS: The three groups of patients did not differ significantly in their demographics (age, gender and laterality), donor endothelial cell counts, preoperative visual acuity or intraocular pressure (IOP). At 12 months postoperatively, BCVA was similar in the three groups, as was the mean spherical equivalent and cylinder. The time to sutures removal was significantly shorter in the HTH PK versus regular PK groups (3.8 (1.2) vs 9.7 (1.1) months, p<0.0001), and the endothelial cell counts were significantly higher (p = 0.003). The IOP was higher in the HTH PK patients than in regular PK patients (p = 0.04). All high-order aberrations tested were significantly higher in the HTH PK than in the regular PK groups (p<0.01). Regular PK had a higher rate of astigmatism treated with relaxing incisions (n = 7 vs n = 2 in HTH PK) and dehiscence of wound incision (n = 2, versus n = 0 in HTH PK). CONCLUSIONS: BCVA and refractive results are similar after half-top-hat, top-hat and regular PK. Half-top-hat PK substantially speeds up visual recovery and contributes to significantly higher endothelial cell counts in the grafts 1 year after surgery.

Posterior lamellar keratoplasty--comparison of deep lamellar endothelial keratoplasty and Descemet stripping automated endothelial keratoplasty in the same patients: a patient's perspective.

AIM: To evaluate patients' perspectives on endothelial keratoplasty and to compare the outcomes of deep lamellar endothelial keratoplasty (DLEK) and Descemet stripping automated endothelial keratoplasty (DSAEK), performed in the same patients. METHODS: A fellow eye, comparative retrospective case series. The records of 14 patients (28 eyes) who underwent DLEK in one eye and DSAEK surgery in their fellow eye between 2003 and 2007 were reviewed. Two patients were excluded from the study. Both these techniques were compared for intra- and postoperative complications, visual and refractive outcomes including higher-order ocular aberrations (HOA). Patient satisfaction for both procedures was prospectively evaluated using a subjective questionnaire. RESULTS: Nine (75%) of the 12 patients perceived better vision in the DSAEK operated eye. Eight (66.6%) of the patients reported faster recovery following DSAEK. Ten (83%) of them preferred the outcomes of the DSAEK surgery. The intra- and postoperative complications were comparable between both procedures. There was no significant difference in visual outcomes between the procedures. However, the DLEK procedure was associated with a significantly higher degree (p<0.05) of HOA. Endothelial cell loss was similar following DLEK and DSAEK. CONCLUSIONS: We conclude that most patients prefer the DSAEK operation, although there are no differences in visual outcomes between DLEK and DSAEK. Avoidance of surgery-induced hyperopia and HOA is the main benefit of the DSAEK technique.

Femtosecond laser versus manual dissection for top hat penetrating keratoplasty.

AIM: To compare the outcomes of IntraLase-enabled top hat penetrating keratoplasty (IEK) versus retrospective results of manual top hat penetrating keratoplasty (TH-PKP) and conventional PKP. PATIENTS/METHODS: This non-randomised prospective study included 94 eyes: 23 eyes underwent IEK, 36 TH-PKP and 35 conventional PKP. Preoperative and postoperative manifest refraction, uncorrected and best-spectacle corrected visual acuity (BSCVA), high-order ocular aberrations (HOA), endothelial cell counts and complications were analysed. RESULTS: At 12 months of follow-up, the mean log MAR BSCVA was 0.32 (SD 0.31) in the IEK group, 0.53 (0.36) in the TH PKP group (p = 0.03) and 0.39 (0.30) in the conventional PKP group (p = 0.4). The mean spherical equivalent was similar between the groups and was less than -2.2 dioptres. The mean cylinder was similar in the IEK and conventional PKP group (3.6 (1.9) dioptres and 4.1 (1.8) dioptres, respectively), and was significantly lower than the TH-PKP group (5.1 (3.2) dioptres, p = 0.04). The complications rate and high-order ocular aberrations were similar between the three groups studied. The mean endothelial cell loss was significantly lower at 12 months of follow-up in the IEK and the TH-PKP groups versus conventional PKP (32.4% and 22.3% vs 40.8%, respectively) (p = 0.05). The mean time to suture removal was 4.1 (1.2) months in the IEK group and 3.9 (1.5) months in the TH-PKP group versus 9.7 (1.1) months in the conventional PKP group (p<0.0001). CONCLUSIONS: IEK is a safe and stable procedure. It results in higher endothelial counts and faster suture removal in comparison with the conventional PKP, and has less astigmatism and better BSCVA in comparison with the manual TH-PKP.

[Comparison of different antifungal treatment regimens for experimental Candida endophthalmitis in rabbit models]. / Tavsanlarda olusturulan deneysel Candida endoftalmisinde farkli tedavi seçeneklerinin karsilastirilmasi.

The aim of this study was to evaluate the treatment options of experimental in-vivo Candida endophthalmitis. For inoculation, a 0.1 ml of suspension of Candida albicans was injected into the vitreous of the right eye of each New Zealand rabbit. On the 15th day, the clinical evaluation for the resultant endophthalmitis was noted, and vitreous samples were obtained. On the 21st day, culture positive eyes were divided into four groups in terms of treatment modalities. Group 1 (n = 7) received intravitreal amphotericin B injection, group 2 (n = 8) received both intravitreal dexamethasone and amphotericin B injections, group 3 (n = 8) underwent pars plana vitrectomy (PPV) and amphotericin B injection, and group 4 (n = 8) underwent PPV and both amphotericin B and silicone oil injections. The vitreous samples obtained from right eyes of the rabbits on the 15th day, were all culture positive for Candida albicans. On the 35th day, the least colony counts (colony forming unit) were present in eyes that received only intravitreal amphotericin B injection in group 1, followed by group 4 that underwent PPV and both amphotericin B and silicone oil injections. In Candida endophthalmitis, intravitreal injection of amphotericin B without steroid appears to be the primary choice of therapy. In cases who fail to respond to this regimen alone, PPV in combination with silicone oil injection may be considered. Benefit-risk ratio should be cautiously interpreted for application of intravitreal steroid injection.

Corneal wound malapposition after penetrating keratoplasty: an optical coherence tomography study.

AIMS: To examine the wound configuration after penetrating keratoplasty (PKP) using anterior segment optical coherence tomography (OCT). METHODS: All PKP patients who were examined for routine follow-up, between November and December 2006, after having all their sutures removed were included. Patients underwent clinical examination, refraction, corneal topography, aberrometry and Visante anterior segment OCT. RESULTS: 204 graft-host sections from 27 eyes (25 patients, mean age 51.6 (SD 17.7) years) were analysed. Although all the graft-host junctions had continuous smooth epithelial surfaces, 124 of them (60.8%) had internal graft-host malappositions such as gapes (15.7%), steps (30%) or protrusions (15.2%). Keratoconus patients had significantly more graft steps (p<0.05) while those transplanted for endothelial dysfunctions had more protrusions (p<0.01). Graft oversizing significantly increased the size of malappositions. Internal gapes or steps significantly reduced the graft-host touch. Intraocular pressure (IOP), final refraction and final keratometric cylinder were all significantly correlated with the presence and size of the malapposition, while steeper keratometry and tilt aberrations correlated with diminished graft-host touch. CONCLUSIONS: After PKP, internal graft-host malapposition is relatively common and associated with increased ametropia, astigmatism, IOP and optical tilt aberrations.

Structure and dynamic regulation of Src-family kinases.

Src-family kinases are modular signaling proteins involved in a diverse array of cellular processes. All members of the Src family share the same domain organization, with modular SH3, SH2 and kinase domains followed by a C-terminal negative regulatory tail. X-ray crystallographic analyses of several Src family members have revealed critical roles for the SH3 and SH2 domains in the down-regulation of the kinase domain. This review focuses on biological, biophysical, and computational studies that reveal conformationally distinct active states within this unique kinase family.

Half-top-hat--a new wound configuration for penetrating keratoplasty.

AIMS: To describe a new, simpler method of achieving the advantages of a lamellar wound configuration in penetrating keratoplasty (PKP)--the half-top-hat (HTH) configuration. METHODS: The donor corneal button was prepared in a top-hat configuration, as previously described. It consisted of a central, full-thickness part, 7-8 mm in diameter, surrounded by a peripheral lamellar wing of deep stroma and endothelium that was 0.5 mm in width (wing diameter 8-9 mm). The recipient bed was prepared by a straight full thickness trephination with a suction trephine. The donor button was positioned by sliding the peripheral wing under the recipient bed. Sixteen 10-0 interrupted sutures and a single continuous 16-bite 11-0 nylon sutures were placed. The interrupted sutures were passed so as to go through the wing, in order to ensure a good apposition of the wing to the inner corneal surface of the recipient. Anterior segment optical coherence tomography (Visante OCT) was used to image the position and alignment of the corneal graft postoperatively. RESULTS: Ten eyes of 10 patients (mean (SD) age: 73.7 (11.4) years, 7 females) underwent HTH PKP. The donor lenticule diameter was 7.7 (0.3) mm (wing diameter 8.7 (0.3) mm). The mean follow-up time was 2.4 (0.7) months. The last median visual acuity was 20/200 (range 20/80-counting fingers), and the last IOP was 18.2 (8.8) mm Hg. No major intraoperative complications were noted. No postoperative events of graft rejection were documented. No anterior surface misalignment was noted either clinically or by OCT. One patient had a rise in IOP postoperatively. CONCLUSIONS: Half-top-hat wound configuration is a valid alternative in penetrating keratoplasty. Its advantages include better apposition of donor and recipient corneas, improved tectonic strength to prevent graft dehiscence, the possibility of early sutures removal and being a simpler procedure to perform.

Serum cytokine levels in active uveitis and remission.

Serum levels of interleukin(IL)-8, IL-6, and (TNF)-alpha were measured in 25 patients during active uveitis and uveitis in remission and compared to age-matched controls. Levels of IL-8 and IL-6 were significantly elevated in patients with active disease and were decreased during remission. IL-8 levels were highest in patients with anterior uveitis, with greatest difference between active disease and remission. No consistent pattern was observed for TNF-alpha. In conclusion, serum cytokine levels are elevated in active noninfectious uveitis. The rise in IL-8 may suggest innate immune mechanisms in the acute disease, while IL-6 participates in modulation of inflammation in the chronic disease.