Investigating the role connective tissue fibroblasts play in the altered muscle anatomy associated with the limb abnormality, Radial Dysplasia.

Radial dysplasia (RD) is a congenital upper limb birth defect that presents with changes to the upper limb anatomy, including a shortened or absent radius, bowed ulna, thumb malformations, a radially deviated hand and a range of muscle and tendon malformations, including absent or abnormally shaped muscle bundles. Current treatments to address wrist instability caused by a shortened or absent radius frequently require an initial soft tissue distraction intervention followed by a wrist stabilisation procedure. Following these surgical interventions, however, recurrence of the wrist deviation remains a common, long-term problem following treatment. The impact of the abnormal soft connective tissue (muscle and tendon) anatomy on the clinical presentation of RD and the complications following surgery are not understood. To address this, we have examined the muscle, fascia and the fascial irregular connective tissue (ICT) fibroblasts found within soft connective tissues, from RD patients. We show that ICT fibroblasts isolated from RD patients are functionally abnormal when compared to the same cells isolated from control patients and secrete a relatively disordered extracellular matrix (ECM). Furthermore, we show that ICT fibroblast dysfunction is a unifying feature found in RD patients, even when the RD clinical presentation is caused by distinct genetic syndromes.

Fetal Cystic Lymphatic Malformations: Systematic Review on Pregnancy and Neonatal Outcomes.

OBJECTIVE: Evaluate pregnancy and neonatal outcomes with fetal cystic lymphatic malformations (LMs), excluding those arising from the posterior neck, to facilitate patient counseling. METHOD: A systematic review was performed in accordance with PRISMA guidance. Case series and case reports published between 2000 and 2022 were included. RESULTS: Sixty-five studies (96 fetuses) met the inclusion criteria. The average gestational age at diagnosis was 25.5 weeks with the commonest location being the anterior neck (28%). All patients were diagnosed with LM using two-dimensional (2D) ultrasound. Prenatal progression in LM size, presence of intralesional bleeding, or fetal hydrops occurred in 70% (41/59), 9% (5/59), and 3% (2/59), respectively. Chromosomal and structural abnormalities were reported in 4% (2/52) and 2% (2/96), respectively. Overall livebirth rate was 94% (79/84); 12/96 resulted in termination and 5/84 in in utero demise. The average gestational age of delivery was 37.7 weeks. Exactly 19% (15/79) had a vaginal birth, of which shoulder dystocia occurred in one infant. Ex utero intrapartum treatment (EXIT) procedure was performed in 13% (10/79). Postnatal treatment commonly involved surgical excision 38% (30/79), sclerotherapy in 21.5% (17/79), or combination of both in 11.4% (9/79). Of those with reported follow-up, 4 died within 1 year, 1 developed heart failure at 2 years of life, and the remaining 44 had normal developmental outcomes. CONCLUSION: Fetal cystic LMs, excluding those in the posterior neck, are not commonly associated with chromosomal, or additional structural abnormalities. They usually increase in size before delivery with only a minority developing complications. The good developmental outcome was reported in all survivors.

Population-based cancer incidence and mortality rates and ratios among adults with intellectual disabilities in Scotland: a retrospective cohort study with record linkage.

: Objective : To provide contemporary data on cancer mortality rates within the context of incidence in the population with intellectual disabilities. : Methods : Scotland's 2011 Census was used to identify adults with intellectual disabilities and controls with records linked to the Scottish Cancer Registry and death certificate data (March 2011-December 2019). The control cohort without intellectual disabilities and/or autism were used for indirect standardisation and calculation of crude incident rates/crude mortality rates, and age-sex standardised incident rate ratios/standardised mortality ratios (SIR/SMR), with 95% CIs. : Results : Adults with intellectual disabilities were most likely diagnosed cancers of digestive, specifically colorectal (14.2%), lung (9.3%), breast (female 22.9%), body of the uterus (female 9.3%) and male genital organs (male 17.6%). Higher incident cancers included metastatic cancer of unknown primary origin (female SIR=1.70, male SIR=2.08), body of uterus (female SIR=1.63), ovarian (female SIR=1.59), kidney (female SIR=1.85) and testicular (male SIR=2.49). SMRs were higher, regardless of a higher, similar or lower incidence (female SMR=1.34, male SMR=1.07). Excess mortality risk was found for colorectal (total SMR=1.54, male SMR=1.59), kidney (total SMR=2.01 u, female SMR=2.85 u), female genital organs (SMR=2.34 (ovarian SMR=2.86 u, body of uterus SMR=2.11), breast (female SMR=1.58) and metastatic cancer of unknown primary origin (female SMR=2.50 u, male SMR=2.84). : Conclusions : Adults with intellectual disabilities were more likely to die of cancer than the general population. Reasons for this may include later presentation/diagnosis (so poorer outcomes), poorer treatment/compliance or both. Accessible public health approaches are important for people with intellectual disabilities, and healthcare professionals need to be aware of the different cancer experiences faced by this population.

Study protocol: the ILANA study - exploring optimal implementation strategies for long-acting antiretroviral therapy to ensure equity in clinical care and policy for women, racially minoritised people and older people living with HIV in the UK - a qualitative multiphase longitudinal study design.

INTRODUCTION: Cabotegravir and rilpivirine (CAB+RPV long-acting (LA)) is recommended as a treatment for HIV-1 allowing people living with HIV to receive 2 monthly injectable treatment, rather than daily pills. Providing injectable therapy in a system designed to provide and manage study participants on oral treatments poses logistical challenges namely how resources are used to accommodate patient preference within constrained health economies with capacity limitations. In this pragmatic multicentre study, we aim to understand the implementation of CAB-RPV-LA administration in two settings via mixed methods to explore perspectives of participants and the clinical team delivering CAB+RPV LA. METHODS AND ANALYSIS: Women, racially minoritised people and older people are chronically under-represented in HIV clinical trials so the ILANA trial has set recruitment caps to ensure recruitment of 50% women, 50% ethnically diverse people and 30% over 50 years of age to include a more representative study population. Using a mixed-methods approach, the primary objective is to identify and evaluate the critical implementation strategies for CAB+RPV LA in both hospital and community settings. Secondary objectives include evaluating feasibility and acceptability of CAB+RPV LA administration at UK clinics and community settings from the perspective of HIV care providers, nurses and representatives at community sites, evaluating barriers to implementation, the utility of implementation strategies and adherence. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Health Research Authority Research Ethics Committee (REC reference: 22/PR/0318). The dissemination strategy has been formulated with the SHARE Collaborative Community Advisory Board to maximise the impact of this work on clinical care and policy. This strategy draws on and leverages existing resources within the participating organisations, such as their academic infrastructure, professional relationships and community networks. The strategy will leverage the Public Engagement Team and press office to support dissemination of findings. TRIAL REGISTRATION NUMBER: NCT05294159.

Specific fluorine labeling of the HyHEL10 antibody affects antigen binding and dynamics.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

Effects of combined deferiprone with deferoxamine on right ventricular function in thalassaemia major.

BACKGROUND: Combination therapy with deferoxamine and oral deferiprone is superior to deferoxamine alone in removing cardiac iron and improving left ventricular ejection fraction (LVEF). The right ventricle (RV) is also affected by the toxic effects of iron and may cause additional cardiovascular perturbation. We assessed the effects of combination therapy on the RV in thalassaemia major (TM) using cardiovascular magnetic resonance (CMR). METHODS: We retrieved imaging data from 2 treatment trials and re-analyzed the data for the RV responses: Trial 1 was a randomized controlled trial (RCT) of 65 TM patients with mild-moderate cardiac siderosis receiving combination therapy or deferoxamine with placebo; Trial 2 was an open label longitudinal trial assessing combination therapy in 15 TM patients with severe iron loading. RESULTS: In the RCT, combination therapy with deferoxamine and deferiprone was superior to deferoxamine alone for improving RVEF (3.6 vs 0.7%, p = 0.02). The increase in RVEF was greater with lower baseline T2* 8-12 ms (4.7 vs 0.5%, p = 0.01) than with T2* 12-20 ms (2.2 vs 0.8%, p = 0.47). In patients with severe cardiac siderosis, substantial improvement in RVEF was seen with open-label combination therapy (10.5% ± 5.6%, p < 0.01). CONCLUSIONS: In the RCT of mild to moderate cardiac iron loading, combination treatment improved RV function significantly more than deferoxamine alone. Combination treatment also improved RV function in severe cardiac siderosis. Therefore adding deferiprone to deferoxamine has beneficial effects on both RV and LV function in TM patients with cardiac siderosis.

Surgical intervention for congenital nail fold hypertrophy.

Congenital nail fold hypertrophy of the hallux is an uncommon abnormality affecting the periungual soft tissue of the great toe. It is usually identified at birth or shortly thereafter, and is known to spontaneously resolve in most cases. In this report, we describe the case of a 14-month-old boy presenting with nail fold hypertrophy of both great toes. The completely united skin bridge covering the nail on the right was excised and the nail folds recreated, with debulking of the left hypertrophic nail fold. We propose that management should be conservative in the first instance and that surgery should be reserved for cases in which 1) inflammation is unresponsive to conservative measures, 2) there is a dense condensation of tissue crossing the nail surface, or 3) there is significant hypertrophy persisting past 1 year of age with no signs of resolution.

The benefits of a ward simulation exercise as a learning experience.

This article describes the findings from a pilot study undertaken to identify the potential benefits of a ward simulation exercise in developing the capabilities of newly qualified nurses. Eight newly qualified nurses were recruited to participate in this pilot study which was based in the Clinical Skills Centre, Ninewells Hospital, Dundee. This pilot study was performed in conjunction with NHS Tayside Practice Education Facilitators and the University of Dundee. Data collection methods involved reflective learning logs which were reviewed independently by an expert group of teachers and practitioners. A focus group session was also undertaken to understand the lived experience of the newly qualified nurse during the ward simulation exercise. Core themes (listed in order of importance) related to the professional development of newly qualified nurses that were identified through this pilot study were: an increase in confidence, development of stress management skills, improved management of the acutely unwell patient, the transfer of skills learnt in simulation to the clinical setting, development of communication skills and reflection skills. Participants in this pilot study demonstrated increased levels of confidence in their communication skills, their ability to prioritize care and to engage in collaborative teamworking.

Fanconi Anemia: Examining Guidelines for Testing All Patients with Hand Anomalies Using a Machine Learning Approach.

BACKGROUND: This study investigated the questionable necessity of genetic testing for Fanconi anemia in children with hand anomalies. The current UK guidelines suggest that every child with radial ray dysplasia or a thumb anomaly should undergo further cost intensive investigation for Fanconi anemia. In this study we reviewed the numbers of patients and referral patterns, as well as the financial and service provision implications UK guidelines provide. METHODS: Over three years, every patient with thumb or radial ray anomaly referred to our service was tested for Fanconi Anemia. CART Analysis and machine learning techniques using Waikato Environment for Knowledge Analysis were applied to evaluate single clinical features predicting Fanconi anemia. RESULTS: Youden Index and Predictive Summary Index (PSI) scores suggested no clinical significance of hand anomalies associated with Fanconi anemia. CART Analysis and attribute evaluation with Waikato Environment for Knowledge Analysis (WEKA) showed no single feature predictive for Fanconi anemia. Furthermore, none of the positive Fanconi anemia patients in this study had an isolated upper limb anomaly without presenting other features of Fanconi anemia. CONCLUSION: As a conclusion, this study does not support Fanconi anemia testing for isolated hand abnormalities in the absence of other features associated with this blood disease.

Design of novel and potent cPLA2α inhibitors containing an α-methyl-2-ketothiazole as a metabolically stable serine trap.

We report the design of novel, potent cPLA(2)α inhibitors that possess an α-methyl-2-ketothiazole that acts as a serine-reactive moiety. We describe the optimization of the series for potency and metabolic stability towards ketone reduction. This was achieved by attenuating the reactivity of the ketone using a combination of electronic and steric effects.

Relation of myocardial T2* to right ventricular function in thalassaemia major.

AIMS: Myocardial T2* cardiovascular magnetic resonance (CMR) provides a rapid and reproducible measure of cardiac iron loading and is being increasingly used worldwide for monitoring of transfusion-dependent thalassaemia patients. Although myocardial siderosis (T2* <20 ms) is associated with impaired left ventricular (LV) function, little is known of its relation with right ventricular (RV) function. The aim of this study was to investigate the relationship between cardiac T2* and RV function. METHODS AND RESULTS: A retrospective analysis of 319 patients with beta-thalassaemia major presenting for their first CMR scan was performed (45.1% male, mean age 25.6 years). In patients with normal myocardial T2* (>20 ms), the RV ejection fraction (EF) was within the normal range in 98% of patients. When myocardial T2* was <20 ms, there was a progressive and significant decline in RV EF. There was a linear relationship between RV and LV EF. CONCLUSION: Myocardial iron deposition is strongly associated with RV dysfunction, which mirrors the decrease in LV function seen with worsening cardiac iron loading. Right ventricular dysfunction may play a significant role in heart failure associated with myocardial siderosis.

Correction of Thumb Duplication: A Systematic Review of Surgical Techniques.

Surgical intervention for thumb duplication can be divided into three categories: simple excision of the accessory thumb, excision of the accessory thumb with reconstruction from available "spare parts," and combining the two thumbs into one, as described by Bilhaut. This prospectively PROSPERO registered systematic review evaluates the overall, aesthetic and functional outcomes for the latter two options (reconstruction from spare parts vs. combining two thumbs into one), aiming to facilitate evidence-based decision making when addressing thumb duplication and direct future research. The review was performed in accordance with the Cochrane Handbook of Systematic Reviews and PRISMA statement. Embase, PubMed, Medline, and Cochrane databases were systematically searched. Studies offering comparisons of techniques were included. Risk of bias was assessed using the Risk of Bias In Non-randomized Studies-of Intervention tool. The quality of the evidence was assessed using the Grading of Recommendations, Assessment, Development, and Evaluation. Ten retrospective observational studies were included. Data did not consistently allow analysis by procedure type. Four studies reported similar overall outcomes between techniques, while two specifically reported poor overall outcomes for the Bilhaut procedure. Two studies reported comparatively worse aesthetic outcomes for the Bilhaut procedure with four studies reporting comparatively improved functional outcomes for this procedure. Overall, interpretation of outcomes was challenging with no patient-reported outcome measures used. The quality of the evidence was universally "very low" due to all studies being at risk of methodological bias. Based on the available evidence, surgical techniques for thumb duplication correction appear comparable regarding overall outcome. There is limited evidence suggesting reconstruction with spare parts offers superior aesthetic outcomes at the expense of stability. The level of evidence is III.

Association energetics of cross-reactive and specific antibodies.

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.

RADIATE - Radial Dysplasia Assessment, Treatment and Aetiology: protocol for the development of a core outcome set using a Delphi survey.

BACKGROUND: Radial dysplasia (RD) is a disfiguring, potentially disabling congenital upper limb anomaly. Multiple surgical techniques are in current use, with little agreement on the optimal treatment approach. At present, no core outcome set exists specifically for RD, and the literature is dominated by retrospective case series. A recent systematic review by this group demonstrated significant heterogeneity on which outcomes are measured and how they are measured. METHODS/DESIGN: The RADIATE study will conduct a three-round online Delphi process, involving adult RD patients, the parents of children with RD, hand surgeons and hand therapists. The initial list of outcomes was drawn from our recent systematic review and will be supplemented by suggestions from the stakeholder groups. Following the Delphi process, outcomes that meet the consensus in definition will be ratified at a final consensus meeting. We will then follow the COSMIN guidelines to select outcome measurement instruments. Where appropriate, these will overlap with the outcome measures specified in the forthcoming standard set for congenital upper limb anomalies published by the International Consortium for Health Outcomes Measurement. DISCUSSION: The Radial Dysplasia Assessment, Treatment and Aetiology (RADIATE) study aims to address the uncertainty in the treatment of RD, and to begin to answer the question 'What is the most appropriate treatment of the forearm and hand for children with RD?' by establishing a core outcome set. TRIAL REGISTRATION: COMET initiative study, 902 . Registered in May 2016.

The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in diabetic retinopathy.

PURPOSE: The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy. METHODS: Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis. RESULTS: Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2. CONCLUSIONS: These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.

Student midwives' experiences in relation to assessment of maternal postnatal genital tract health: A case study analysis.

OBJECTIVE: to explore student midwives' experiences of postnatal genital tract assessment within midwifery preregistration curricula. DESIGN: a single, instrumental case study design was employed involving final year student midwives. Ethical approval was gained from the Higher Education Institution at the data collection site. Sampling was purposeful and data were collected using a survey (n = 25); narrative style in depth interviews (n = 11), review of programme documentation and a student midwife / researcher data workshop. SETTING: one Higher Education Institution in the north of England. FINDINGS: three themes were identified from the data analysis, awareness of assessment methods, accessing learning opportunities and actualisation of learning. The awareness theme highlights that most students were aware of potential signs and symptoms associated with genital tract assessment and health however; difficulties were identified concerning assessment of lochia, encountering sequential assessments and recognising potential for deterioration. This awareness was influenced by access to practice based learning opportunities. Access differed due to variation in postnatal provision, service pressures and variety in mentor practices regarding selecting and creating learning opportunities. This study suggests actualisation of learning and confidence in genital tract assessment was achieved when opportunities to integrate theory and practice occurred. Actualisation was hindered by limited allocation of curriculum time specifically for postnatal maternal assessment content and assessment strategies in comparison to other aspects of midwifery knowledge. CONCLUSIONS: student midwives' experiences, awareness and learning actualisation varied in relation to the development of knowledge and confidence in maternal postnatal genital tract assessment. While clinical and theoretical learning opportunities were available, access and experience varied and limitations were identified. A number of recommendations are outlined to enhance the students learning experiences in practice and HEI settings, which address placement planning, mentor preparation, the student voice and supporting curricula documentation.

Understanding antibody-antigen associations by molecular dynamics simulations: detection of important intra- and inter-molecular salt bridges.

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure-function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize 'coincident epitopes'. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63- HEL reveals the presence of functionally important saltbridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing saltbridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed saltbridges are present in one or more of the other three complexes and/or involve \"hot-spot\" epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The "hot spot" epitope residue Lys97Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this intermolecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed saltbridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97Y in the formation of inter-molecular triad further validates the functional importance of Lys97Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights "hot-spot" molecular interactions in HyHEL63-HEL complex.

Vascular endothelial growth factor C promotes survival of retinal vascular endothelial cells via vascular endothelial growth factor receptor-2.

AIM: To determine vascular endothelial growth factor C (VEGF-C) expression in retinal endothelial cells, its antiapoptotic potential and its putative role in diabetic retinopathy. METHOD: Cultured retinal endothelial cells and pericytes were exposed to tumour necrosis factor (TNF)alpha and VEGF-C expression determined by reverse transcriptase-polymerase chain reaction. Secreted VEGF-C protein levels in conditioned media from endothelial cells were examined by western blotting analysis. The ability of VEGF-C to prevent apoptosis induced by TNFalpha or hyperglycaemia in endothelial cells was assessed by flow cytometry. The expression of VEGF-C in diabetic retinopathy was studied by immunohistochemistry of retinal tissue. RESULT: VEGF-C was expressed by both vascular endothelial cells and pericytes. TNFalpha up regulated both VEGF-C and vascular endothelial growth factor receptor-2 (VEGFR)-2 expression in endothelial cells in a dose-dependent manner, but had no effect on VEGFR-3. Flow cytometry results showed that VEGF-C prevented endothelial cell apoptosis induced by TNFalpha and hyperglycaemia and that the antiapoptotic effect was mainly via VEGFR-2. In pericytes, the expression of VEGF-C mRNA remained stable on exogenous TNFalpha treatment. VEGF-C immunostaining was increased in retinal vessels in specimens with diabetes compared with retinal specimens from controls without diabetes. CONCLUSION: In retinal endothelial cells, TNFalpha stimulates the expression of VEGF-C, which in turn protects endothelial cells from apoptosis induced by TNFalpha or hyperglycaemia via VEGFR-2 and thus helps sustain retinal neovascularisation.

Antibody recognition imaging by force microscopy.

We have developed a method that combines dynamic force microscopy with the simultaneous molecular recognition of an antigen by an antibody, during imaging. A magnetically oscillated atomic force microscopy tip carrying a tethered antibody was scanned over a surface to which lysozyme was bound. By oscillating the probe at an amplitude of only a few nanometers, the antibody was kept in close proximity to the surface, allowing fast and efficient antigen recognition and gentle interaction between tip and sample. Antigenic sites were evident from reduction of the oscillation amplitude, as a result of antibody-antigen recognition during the lateral scan. Lysozyme molecules bound to the surface were recognized by the antibody on the scanning tip with a few nanometers lateral resolution. In principle, any ligand can be tethered to the tip; thus, this technique could potentially be used for nanometer-scale epitope mapping of biomolecules and localizing receptor sites during biological processes.

Magnitude of the hydrophobic effect at central versus peripheral sites in protein-protein interfaces.

Hydrophobic interactions are essential for stabilizing protein-protein complexes, whose interfaces generally consist of a central cluster of hot spot residues surrounded by less important peripheral residues. According to the O-ring hypothesis, a condition for high affinity binding is solvent exclusion from interacting residues. This hypothesis predicts that the hydrophobicity at the center is significantly greater than at the periphery, which we estimated at 21 cal mol(-1) A(-2). To measure the hydrophobicity at the center, structures of an antigen-antibody complex where a buried phenylalanine was replaced by smaller hydrophobic residues were determined. By correlating structural changes with binding free energies, we estimate the hydrophobicity at this central site to be 46 cal mol(-1) A(-2), twice that at the periphery. This context dependence of the hydrophobic effect explains the clustering of hot spots at interface centers and has implications for hot spot prediction and the design of small molecule inhibitors.