Safety and clinical outcomes of omitting same and next day review after DMEK performed with an inferior peripheral iridotomy.

BACKGROUND/OBJECTIVES: To determine the incidence of day one postoperative complications after Descemet Membrane Endothelial Keratoplasty (DMEK) performed with intraoperative inferior peripheral iridotomy (PI), and whether their early detection influences postoperative intervention. SUBJECTS/METHODS: 70 eyes of 70 consecutive patients that underwent DMEK from August 2019 to August 2021 at a single UK centre were retrospectively analysed. Cases that did not have an inferior PI were excluded. Any action taken at day one and week one postoperative review was noted. RESULTS: No pupil block or other major adverse events were found at day one review. At one week, 14 eyes (20%) required re-bubbling, all of which had been fully attached at the day one review. CONCLUSIONS: This series suggests that inferior PI performed alongside DMEK alone or triple DMEK effectively minimizes the risk of pupil block. Since no early complications occurred in this cohort requiring immediate intervention, it may be safe to defer review of these patients to a later time point.

X-ray constrained wavefunctions based on Hirshfeld atoms. I. Method and review.

The X-ray constrained wavefunction (XCW) procedure for obtaining an experimentally reconstructed wavefunction from X-ray diffraction data is reviewed. The two-center probability distribution model used to perform nuclear-position averaging in the original paper [Grimwood & Jayatilaka (2001). Acta Cryst. A57, 87-100] is carefully distinguished from the newer one-center probability distribution model. In the one-center model, Hirshfeld atoms are used, and the Hirshfeld atom based X-ray constrained wavefunction (HA-XCW) procedure is described for the first time, as well as its efficient implementation. In this context, the definition of the related X-ray wavefunction refinement (XWR) method is refined. The key halting problem for the XCW method - the procedure by which one determines when overfitting has occurred - is named and work on it reviewed.

X-ray constrained wavefunctions based on Hirshfeld atoms. II. Reproducibility of electron densities in crystals of α-oxalic acid dihydrate.

The Hirshfeld atom-based X-ray constrained wavefunction fitting (HA-XCW) procedure is tested for its reproducibility, and the information content of the fitted wavefunction is critically assessed. Fourteen different α-oxalic acid dihydrate data sets are used for this purpose, and the first joint fitting to 12 of these data sets is reported. There are systematic features in the electron density obtained from all data sets which agree with higher level benchmark calculations, but there are also many other strong systematic features which disagree with the reference calculations, most notably those associated with the electron density near the nuclei. To enhance reproducibility, three new protocols are described and tested to address the halting problem of XCW fitting, namely: an empirical power-function method, which is useful for estimating the accuracy of the structure factor uncertainties; an asymptotic extrapolation method based on ideas from density functional theory; and a `conservative method' whereby the smallest value of the regularization parameter is chosen from a series of data sets, or subsets.

Selective Laser Trabeculoplasty for Steroid-Induced Ocular Hypertension following Endothelial Keratoplasty.

Purpose: To examine the safety and efficacy of selective laser trabeculoplasty (SLT) in patients responding to topical steroids with elevated intraocular pressure following endothelial keratoplasty.Methods: Patients that underwent Descemet Membrane Endothelial Keratoplasty (DMEK) or Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) were offered SLT as first-line therapy if they presented with steroid response and met inclusion criteria. Patient demographics, best-corrected visual acuity (BCVA), steroid and glaucoma regimens were recorded before and after SLT.Results: Twelve eyes of eight patients were recruited to the study. All patients demonstrated a reduction in IOP and only one patient remained on anti-glaucoma drops after SLT. The mean (±SD) reduction in IOP following SLT was 8.8 (±4.9) mmHg (p < 0.0001). BCVA remained stable and no adverse events following treatment were observed.Conclusions: SLT may be a safe and effective treatment option that reduces topical antihypertensive burden while allowing continuance of corticosteroids in steroid-responsive eyes at high risk of rejection following endothelial keratoplasty. Larger randomized studies are needed to compare SLT efficacy with topical medication in this patient group and to review any impact on graft survival and glaucoma progression.

Early Endothelialization of Ab Interno Stromal Tectonic Patch in the Management of Corneal Perforation Secondary to Bacterial Keratitis.

PURPOSE: The purpose of this study was to describe a novel surgical technique using an ab interno stromal patch to treat corneal perforation and to present the histological findings of the patch after its removal during definitive mushroom keratoplasty. METHODS: The endothelium had already been removed with the submerged cornea using backgrounds away (SCUBA) technique. RESULTS: The patient was managed successfully with a sutureless ab interno stromal tectonic patch. Two months later, definitive mushroom keratoplasty was performed and the patch was sent for histological examination. Immunohistochemistry revealed a reactive endothelium covering the posterior surface of the graft. Nine months later, her best-corrected visual acuity was 6/9. CONCLUSIONS: This is the first case to our knowledge demonstrating that stromal tectonic grafts without the endothelium can successfully attach to the host tissue and seal a perforation. The stroma may undergo reendothelialization and begin to restore vision, even before penetrating keratoplasty.

Heavy silicone oil tamponade: a multicentre experience.

OBJECTIVE: To report multicentred use of the heavy silicone oil Densiron 68 for anatomical reattachment following rhegmatogenous retinal detachment (RRD) repair and its associated complications. METHODS AND ANALYSIS: Patients from seven vitreoretinal units within the UK that underwent RRD repair with Densiron 68 between January 2015 and December 2019 were identified. Primary outcome measures were primary and final reattachment rate, retained Densiron and failure rate. Secondary outcome measures were duration of tamponade, final visual acuity (VA) and complications of heavy silicone oil. RESULTS: 134 eyes of 134 patients were involved in the study. Primary surgical success was achieved in 48.5%, while a final reattachment rate of 73.4% was observed. The mean duration of Densiron 68 tamponade was 139.5 days. Mean final VA was 1.01 (range 0-2.9). 8 eyes (6.0%) required long-term topical steroids for anterior uveitis, whereas none of the eyes required long-term pressure-lowering treatment. Emulsification rate was 10.7% (6 eyes). CONCLUSION: This is the largest real-world study on Densiron 68 in the UK. Densiron 68 facilitates tamponade of inferior retinal pathology and may be considered as an option for tamponade of inferior retinal pathologies.

Rational antibody design for undruggable targets using kinetically controlled biomolecular probes.

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

Solitary Conjunctivocorneal Plasmacytoma Arising After Cataract Surgery.

PURPOSE: To report a case of solitary plasmacytoma of the conjunctiva with corneal encroachment and to review the literature of all published cases. METHODS: A 70-year-old man presented with 1-month history of pain and watering in the right eye, 7 months after undergoing cataract surgery in the same eye. RESULTS: The mass was surgically excised. Pathology and immunohistochemistry were consistent with plasmacytoma. Staging studies found no evidence of systemic disease. CONCLUSIONS: To our knowledge, this is the first case of plasmacytoma in the literature to involve the cornea. This case was not associated with systemic disease. Surgical excision and brachytherapy remain options for cases without corneal involvement.

fragHAR: towards ab initio quantum-crystallographic X-ray structure refinement for polypeptides and proteins.

The first ab initio aspherical structure refinement against experimental X-ray structure factors for polypeptides and proteins using a fragmentation approach to break up the protein into residues and solvent, thereby speeding up quantum-crystallographic Hirshfeld atom refinement (HAR) calculations, is described. It it found that the geometric and atomic displacement parameters from the new fragHAR method are essentially unchanged from a HAR on the complete unfragmented system when tested on dipeptides, tripeptides and hexapeptides. The largest changes are for the parameters describing H atoms involved in hydrogen-bond interactions, but it is shown that these discrepancies can be removed by including the interacting fragments as a single larger fragment in the fragmentation scheme. Significant speed-ups are observed for the larger systems. Using this approach, it is possible to perform a highly parallelized HAR in reasonable times for large systems. The method has been implemented in the TONTO software.

Proteotyping: Proteomic characterization, classification and identification of microorganisms--A prospectus.

Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels.

Detection of ligand-receptor binding using microfluidic frontal affinity chromatography on proteoliposomes derived directly from native cell membranes.

A method for characterization of ligand binding to membrane receptors in their native cell membrane is presented. The methodology is based on microfluidic frontal affinity chromatography coupled to mass spectrometry (FAC-MS). Proteoliposomes with receptor of interest are prepared directly from cell membranes and serve as a stationary phase in a microfluidic flow cell for frontal analysis. The G-Protein-Coupled Receptor (GPCR) Ste2 involved in the pheromone-induced yeast mating pathway is used as a model receptor for proof of principle characterization. The ligand affinity of the natural pheromone peptide, the α-factor, is compared to a set of pheromone analogs having different receptor affinities. With short preparation time, preserved lipid composition and the ability to immobilize proteoliposomes from any cell membrane, we propose that our methodology with immobilized proteoliposomes together with microfluidics FAC-MS can be an important improvement for ligand-receptor studies in native membranes.

Combining phenotypic and proteomic approaches to identify membrane targets in a 'triple negative' breast cancer cell type.

BACKGROUND: The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a 'triple negative' breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. METHODS: The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other 'triple negative' breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. RESULTS: All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other 'triple negative' breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model. CONCLUSIONS: This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.

Microfluidic flow cell for sequential digestion of immobilized proteoliposomes.

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 µg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 µg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.

Strain-level typing and identification of bacteria using mass spectrometry-based proteomics.

Because of the alarming expansion in the diversity and occurrence of bacteria displaying virulence and resistance to antimicrobial agents, it is increasingly important to be able to detect these microorganisms and to differentiate and identify closely related species, as well as different strains of a given species. In this study, a mass spectrometry proteomics approach is applied, exploiting lipid-based protein immobilization (LPI), wherein intact bacterial cells are bound, via membrane-gold interactions, within a FlowCell. The bound cells are subjected to enzymatic digestion for the generation of peptides, which are subsequently identified, using LC-MS. Following database matching, strain-specific peptides are used for subspecies-level discrimination. The method is shown to enable a reliable typing and identification of closely related strains of the same bacterial species, herein illustrated for Helicobacter pylori .

Membrane protein digestion - comparison of LPI HexaLane with traditional techniques.

Membrane protein profiling and characterization is of immense importance for the understanding of vital processes taking place across cellular membranes. Traditional techniques used for soluble proteins, such as 2D gel electrophoresis, are sometimes not entirely applicable to membrane protein targets, due to their low abundance and hydrophobic character. New tools have been developed that will accelerate research on membrane protein targets. Lipid-based protein immobilization (LPI) is the core technology in a new approach that enables immobilization and digestion of native membrane proteins inside a flow cell format. The presented method is described in the context of comparing the method to traditional approaches where the sample amount that is digested and analyzed is the same.

A giant liposome for single-molecule observation of conformational changes in membrane proteins.

We present an experimental system that allows visualization of conformational changes in membrane proteins at the single-molecule level. The target membrane protein is reconstituted in a giant liposome for independent control of the aqueous environments on the two sides of the membrane. For direct observation of conformational changes, an extra-liposomal site(s) of the target protein is bound to a glass surface, and a probe that is easily visible under a microscope, such as a micron-sized plastic bead, is attached to another site on the intra-liposomal side. A conformational change, or an angular motion in the tiny protein molecule, would manifest as a visible motion of the probe. The attachment of the protein on the glass surface also immobilizes the liposome, greatly facilitating its manipulation such as the probe injection. As a model system, we reconstituted ATP synthase (F(O)F(1)) in liposomes tens of mum in size, attached the protein specifically to a glass surface, and demonstrated its ATP-driven rotation in the membrane through the motion of a submicron bead.

Proteomic analysis of plasma membrane vesicles.

A simple and scalable method is presented for harvesting, purification, and on-chip processing of mammalian plasma membrane vesicles (PMVs) optimized for downstream proteome analysis. After immobilization on a microfluidic flowcell of PMVs, the embedded membrane proteins are proteolytically digested, and the peptides harvested and analyzed by LC-MS/MS. Over 93% of the detected proteins are plasma-membrane-derived.

Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks.

We demonstrate here that nanotube-vesicle networks can be constructed directly from plasma membranes of cultured cells. We used a combination of dithiothreitol (DTT) and formaldehyde to produce micron-sized plasma membrane vesicles that were subsequently shaped into networks using micromanipulation methods previously used on purely synthetic systems. Only a single cell is required to derive material sufficient to build a small network. This protocol covers the advantages of reconstitution in vesicles, such as full control over the solution environment, while keeping the proteins in their original surroundings with the proper orientation. Furthermore, control of membrane protein and lipid content in the networks is achievable by employing different cell types, for example, by overexpression of a desired protein or the use of specialized cell-types as sources for rare proteins and lipids. In general, the method provides simple accessibility for functional studies of plasma membrane constituents. Specifically, it provides a direct means to functionalize nanotube-vesicle networks with desired proteins and lipids for studies of transport activity both across membranes (protein-mediated) and across nanotubes (diffusion), and substrate conversion down to the single-molecule limit. Nanotube-vesicle networks can adopt different geometries and topologies and undergo shape changes at will, providing a flexible system for changing the physical and chemical environment around, for example, a membrane protein. Furthermore, the method offers unique possibilities for extracting membrane and protein material for nanotechnological sensor and analytical devices based on lipid membrane networks.