Corneal Guttae After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: The aim of this study was to report on the occurrence of corneal guttae after Descemet membrane endothelial keratoplasty (DMEK). METHODS: In this retrospective case series, 13 eyes of 13 patients who underwent DMEK at 2 tertiary referral centers between 2007 and 2021 (average available follow-up 73 ± 52 months, range 18-174 months) and showed corneal guttae during postoperative examinations were included. Eye bank images were retrospectively reviewed. RESULTS: Occurrence of guttae was observed by specular microscopy in 13 eyes. In 11 cases, presence of guttae was confirmed by confocal microscopy and in 1 case by histology. Five eyes showed an increase in guttae density during the postoperative course. Surgery indications were Fuchs endothelial corneal dystrophy (n = 11), pseudophakic bullous keratopathy (n = 1), and DMEK graft failure after allograft rejection (n = 1); the latter eye had shown no signs of guttae after primary DMEK. Two eyes with guttae required a repeat DMEK due to graft failure. At the last available follow-up, all 11 remaining eyes had clear corneas and 10 eyes had a best-corrected visual acuity of ≥0.9 (decimal). During donor cornea processing in the eye bank, no guttae were observed on the donor tissue. CONCLUSIONS: Corneal guttae can occur after DMEK including in eyes operated for indications other than Fuchs endothelial corneal dystrophy and most likely guttae were present on the donor graft but were not detectable by routine slit-lamp and light microscopy evaluation in the eye bank. Postoperative guttae density varies among patients and especially small isolated guttae do not seem to affect clinical outcomes.

Long-Term Outcome After Bilateral DMEK for Fuchs Endothelial Corneal Dystrophy.

PURPOSE: The aim of this study was to assess the long-term clinical outcome, complications, and graft survival of bilateral Descemet membrane endothelial keratoplasty (DMEK) in patients with Fuchs endothelial corneal dystrophy. METHODS: This was a retrospective cohort study of 181 patients (362 eyes) with sequential bilateral DMEK for Fuchs endothelial corneal dystrophy. Clinical outcomes were assessed up to 5 years postoperatively. Outcome measures were best-corrected visual acuity, pachymetry, endothelial cell density, graft survival, and complication rates. RESULTS: Contralateral DMEK was performed on average 15 ± 11 months (range: 2-60 months) after the first eye. From 1 until 5 years after DMEK, best-corrected visual acuity, pachymetry, endothelial cell density, and graft survival did not differ between the first and second eyes (all P > 0.05). Graft detachment occurred in 67 eyes (19% [18% first eyes, 19% second eyes], 6% bilateral), graft rejection in 9 eyes (3% [3% first eyes, 2% second eyes], 1% bilateral), glaucoma in 25 eyes (7% [8% first eyes, 6% second eyes], 2% bilateral), and graft failure in 22 eyes (6% [4% first eye, 8% second eye], 2% bilateral). All differences were not significant (all P > 0.05). Five-year graft survival rates were comparable for first and second eyes (0.95 and 0.92, respectively; P = 0.15). CONCLUSIONS: Clinical outcomes after bilateral DMEK are similar in both eyes and sustainable in the longer term. Within the first 5 years, the same complication may rarely occur in the contralateral eye.

Chronically altered ventricular activation causes pro-arrhythmic cardiac electrical remodelling in the chronic AV block dog model.

AIMS: Altered ventricular activation (AVA) causes intraventricular mechanical dyssynchrony (MD) and impedes contraction, promoting pro-arrhythmic electrical remodelling in the chronic atrioventricular block (CAVB) dog. We aimed to study arrhythmogenic and electromechanical outcomes of different degrees of AVA. METHODS AND RESULTS: Following atrioventricular block, AVA was established through idioventricular rhythm (IVR; n = 29), right ventricular apex (RVA; n = 12) pacing or biventricular pacing [cardiac resynchronization therapy (CRT); n = 10]. After ≥3 weeks of bradycardic remodelling, Torsade de Pointes arrhythmia (TdP) inducibility, defined as ≥3 TdP/10 min, was tested with specific IKr-blocker dofetilide (25 µg/kg/5 min). Mechanical dyssynchrony was assessed by echocardiography as time-to-peak (TTP) of left ventricular (LV) free-wall minus septum (ΔTTP). Electrical intraventricular dyssynchrony was assessed as slope of regression line correlating intraventricular LV activation time (AT) and activation recovery interval (ARI). Under sinus rhythm, contraction occurred synchronous (ΔTTP: -8.6 ± 28.9 ms), and latest activated regions seemingly had slightly longer repolarization (AT-ARI slope: -0.4). Acute AV block increased MD in all groups, but following ≥3 weeks of remodelling IVR animals became significantly more TdP inducible (19/29 IVR vs. 5/12 RVA and 2/10 CRT, both P < 0.05 vs. IVR). After chronic AVA, intraventricular MD was lowest in CRT animals (ΔTTP: -8.5 ± 31.2 vs. 55.80 ± 20.0 and 82.7 ± 106.2 ms in CRT, IVR, and RVA, respectively, P < 0.05 RVA vs. CRT). Although dofetilide steepened negative AT-ARI slope in all groups, this heterogeneity in dofetilide-induced ARI prolongation seemed least pronounced in CRT animals (slope to -0.8, -3.2 and -4.5 in CRT, IVR and RVA, respectively). CONCLUSION: Severity of intraventricular MD affects the extent of electrical remodelling and pro-arrhythmic outcome in the CAVB dog model.

First Clinical Experience With Ophthalmic e-Device for Unaided Patient Self-Examination During COVID-19 Lockdown.

PURPOSE: The aim of this study was to describe a new type of medical device that allows for internet-enabled patient self-screening, without the aid of an ophthalmic professional, through biomicroscopy self-imaging and self-measurement of the best-corrected visual acuity (BCVA). METHODS: In this prospective nonrandomized comparative study, 56 patients were instructed to screen their own eyes using a custom-built e-Device containing miniaturized slitlamp optics and a visual acuity Snellen chart virtually projected at 20 ft. BCVA measurements were recorded, and biomicroscopic videos were scored for image quality of the anterior segment status on a scale from 1 to 5 (1 = poor and 5 = excellent) by a blinded observer. RESULTS: After a short instruction, all patients were able to self-image their eyes and perform a self-BCVA measurement using the e-Device. Patient self-image quality with the e-Device scored on average 3.3 (±0.8) for videos (n = 76) and 3.6 (±0.6) for photographs (n = 49). Self-BCVA measurement was within 1 Snellen line from routine BCVA levels in 66 of 72 eyes (92%). When compared with conventional biomicroscopy, patient self-biomicroscopy allowed for recognition of the relevant pathology (or absence thereof) in 26 of 35 eyes (74%); 9 cases showed insufficient image quality attributed to device operating error (n = 6) and mild corneal edema and/or scarring (n = 3). Patient satisfaction with the device was 4.4 (±0.9). CONCLUSIONS: An e-Device for combined BCVA self-measurement and biomicroscopy self-imaging may have potential as an aid in remote ophthalmic examination in the absence of an ophthalmic professional and may be considered for patients who are unable to visit an ophthalmic clinic for routine follow-up.

In Vitro Evaluation of the Feasibility of Slit-Lamp Nd: YAG Laser Descemetorhexis.

PURPOSE: To evaluate in vitro the feasibility and tissue effects of using a slit-lamp neodymium-doped yttrium aluminum garnet (Nd:YAG) laser to create a central descemetorhexis in human donor corneas. METHODS: Twelve human donor corneas ineligible for transplantation were divided into 2 groups, A and B. Group A: 2 "healthy" corneas, which were used to validate the laser parameters; group B: 10 corneas with endothelial guttae, which were used to perform a 4-mm descemetorhexis. Slit-lamp photography, light microscopy, corneal endothelial microscopy, Scheimpflug imaging, optical coherence tomography (OCT) imaging, and histological staining were performed to visualize the efficacy of slit-lamp Nd:YAG laser removal of Descemet membrane and to assess potential tissue damage to the overlying stroma and peripheral endothelium. RESULTS: In all corneas, an Nd:YAG laser 4-mm central descemetorhexis could be consistently performed. The total energy required ranged from 1143 to 2784 mJ. Side effects such as stromal pitting and corneal swelling were observed. CONCLUSIONS: Creating a central descemetorhexis with a slit-lamp Nd:YAG laser proved feasible in vitro. This new technical approach might open the door to a customized in vivo "descemetorhexis-only" treatment for Fuchs endothelial corneal dystrophy eyes, while avoiding the risks associated with intraocular surgery.

Parameters Associated With Endothelial Cell Density Variability After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate which parameters may affect endothelial cell loss after Descemet membrane endothelial keratoplasty (DMEK) by comparing eyes in the low vs high quartile of endothelial cell loss over a follow-up period of 4 years. DESIGN: Retrospective cohort study. METHODS: Donor endothelial cell density (ECD) decline was evaluated for 351 eyes of 275 patients up to 4 years after DMEK for Fuchs endothelial corneal dystrophy (FECD). Eyes with a postoperative endothelial cell loss in the lower quartile at all available follow-up moments were assigned to Group 1 (n = 51) and those in the upper quartile to Group 2 (n = 42). Multinomial regression was used to assess which covariates were related to greater ECD decline. RESULTS: Mean endothelial cell loss as compared to preoperative donor ECD for the entire study group was 33 (±16)%, 36 (±17)%, and 52 (±18)% at 1, 6, and 48 months postoperatively. Endothelial cell loss of Group 1 was 12 (±7)%, 13 (±6)%, and 26 (±8)% at, respectively, 1, 6, and 48 months postoperatively, and 59 (±10)%, 64 (±9)%, and 75 (±5)% in Group 2. Partial graft detachment, donor death cause cardiovascular/stroke (vs cancer), postoperative complications other than graft detachment, and severity of preoperative FECD (all P < .01) showed the strongest relation with greater ECD decline. CONCLUSIONS: DMEK eyes with a completely attached graft and operated in an early stage of FECD may show the lowest endothelial cell loss postoperatively.

Five-Year Graft Survival and Clinical Outcomes of 500 Consecutive Cases After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To report the 5-year graft survival and clinical outcomes after Descemet membrane endothelial keratoplasty (DMEK). METHODS: A retrospective, interventional case series was performed at a tertiary referral center. Five hundred eyes of 393 patients that underwent DMEK for Fuchs endothelial corneal dystrophy, bullous keratopathy, failed previous corneal transplants other than DMEK, or other indications were evaluated for graft survival, best-corrected visual acuity (BCVA), endothelial cell density, postoperative complications, and retransplantation rate. RESULTS: Kaplan-Meier analysis demonstrated an estimated survival probability of 0.90 [95% confidence interval, 0.87-0.94] for the entire cohort at 5 years after DMEK. At this time point, 82% of the eyes achieved a BCVA of ≥20/25 (0.8), 54% achieved ≥20/20 (1.0), and 16% achieved ≥20/17 (1.2). BCVA continued to improve from 6 to 36 months after DMEK surgery (P ≤ 0.005) and then remained stable up to 60 months postoperatively (P > 0.08). Preoperative donor endothelial cell density averaged 2530 (±210) cells/mm and decreased by 37% at 6 months, 40% at 1 year, and 55% at 5 years after DMEK surgery (P < 0.001 between all follow-up time points). During the study period, allograft rejection episodes developed in 2.8% of the eyes, primary graft failure occurred in 0.2%, and secondary graft failure in 2.8% of the eyes. Re-keratoplasty was required in 8.8% of the eyes. CONCLUSIONS: Five-year graft survival after DMEK is high, and visual acuity outcomes remain excellent and are accompanied by a low longer-term complication rate.

Quarter-Descemet Membrane Endothelial Keratoplasty: One- to Two-Year Clinical Outcomes.

PURPOSE: To report clinical outcomes of the first Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) case series performed for central Fuchs endothelial corneal dystrophy. METHODS: This is a prospective, interventional case series analyzing the clinical outcomes of 19 eyes of 19 patients with central Fuchs endothelial corneal dystrophy, that is, with guttae predominantly in the 6- to 7-mm optical zone, who underwent unilateral Quarter-DMEK at a tertiary referral center. Main outcome measures were best-corrected visual acuity (BCVA), endothelial cell density (ECD), and postoperative complications. Included eyes had up to 2 years of postoperative follow-up. RESULTS: At 6 months postoperatively, all eyes reached a BCVA of ≥20/40 (≥0.5): 18 of 19 eyes (95%) with ≥20/25 (≥0.8) and 9 of 19 eyes (42%) with ≥20/20 (≥1.0). Thereafter, BCVA remained stable up to 2 years postoperatively. The mean donor ECD decreased from 2842 ± 139 cells/mm (n = 19) before implantation to 913 ± 434 cells/mm (-68%) at 6 months (n = 19), 869 ± 313 cells/mm (-70%) at 12 months (n = 18), and 758 ± 225 cells/mm (-74%) at 24 months (n = 13) after Quarter-DMEK. Visually significant graft detachment requiring rebubbling occurred in 8 of 19 eyes (42%). CONCLUSIONS: Quarter-DMEK surgery yields visual outcomes similar to those of conventional DMEK and may potentially quadruple the availability of endothelial grafts. Further modifications of the graft preparation and the surgical technique may improve clinical outcomes in terms of lower ECD decrease and fewer graft detachments.

Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) for Fuchs endothelial corneal dystrophy: 6 months clinical outcome.

BACKGROUND/AIM: To assess the clinical outcome of the first series of Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK), a potential hybrid technique between 'descemetorhexis only' and conventional, circular DMEK. METHODS: Prospective interventional case series at a tertiary referral centre. Twelve eyes of 12 patients with central Fuchs endothelial corneal dystrophy underwent Quarter-DMEK, that is, transplantation of one quadrant of a full-diameter DMEK graft, and were evaluated for best-corrected visual acuity (BCVA), endothelial cell density (ECD) and complications up to 6 months postoperatively. RESULTS: At 6 months postoperatively, all eyes reached a BCVA of ≥20/40 (≥0.5), 11/12 (92%) of ≥20/25 (≥0.8) and 6/12 (50%) of ≥20/20 (≥1.0). Mean central ECD decreased from 2867 (±161) cells/mm2 before to 1255 (±514) cells/mm2 at 1 month, 1058 (±455) cells/mm2 at 3 months and 968 (±427) cells/mm2 at 6 months after surgery. Rebubbling was performed in 4/12 eyes (33%) within the first two months. CONCLUSIONS: Quarter-DMEK may be a feasible procedure that allows for visual outcomes similar to conventional, circular DMEK. The relatively large drop in ECD within the first month may have resulted from more extensive endothelial cell migration and/or measurement error (at the graft edges). If longer-term outcomes would resemble those of conventional DMEK, Quarter-DMEK may potentially quadruple the availability of endothelial grafts.

Donor Tissue Preparation for Bowman Layer Transplantation.

PURPOSE: To describe and compare 2 preparation techniques for Bowman layer (BL) grafts for use in BL transplantation. METHODS: A retrospective evaluation of the 2 methods for preparing BL grafts was performed, that is, BL graft preparation from donor globes not eligible for penetrating keratoplasty or endothelial keratoplasty (technique I, n = 36) and BL graft preparation from previously excised corneoscleral buttons (technique II, n = 36) that could not be used for PK or had been denuded of Descemet membrane and endothelium for Descemet membrane endothelial keratoplasty graft preparation. BL graft preparation difficulties were recorded, and the preparation failure rate was examined and compared between the techniques. RESULTS: Overall, BL graft preparation was successful in 51 cases (51/72; 70.8%), of which 25 preparations were successful using technique I, and 26 using technique II, representing a success rate of 69.4% (25/36) and 72.2% (26/36) for techniques I and II, respectively. Reasons for discarding a BL graft were tearing of the BL tissue during the preparation (n = 19) and stroma attached to the graft (n = 2). CONCLUSIONS: Isolated BL grafts can be prepared from both whole donor globes and corneoscleral rims with equivalent success. Preparation from corneoscleral rims may offer the advantage that, from one donor cornea, the posterior layers can be used for Descemet membrane endothelial keratoplasty graft preparation and the anterior part for BL graft preparation.

Calmodulin/CaMKII inhibition improves intercellular communication and impulse propagation in the heart and is antiarrhythmic under conditions when fibrosis is absent.

AIM: In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this. METHODS AND RESULTS: AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P < 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (-20%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 µM) increased CV (6-13%, n= 6, P< 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n= 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n= 4, P< 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes. CONCLUSION: Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias.

Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart.

BACKGROUND: The multifunctional Ca(2+)- and calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is large-scale analysis of phosphorylation events by mass spectrometry. However, current large-scale phosphoproteomics approaches have proved inadequate for high-fidelity identification of kinase-specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue. METHODS AND RESULTS: To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial-delimited expression of the specific peptide inhibitor of CaMKII (AC3-I) or an inactive control (AC3-C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of ß-adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3-I and AC3-C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high-resolution LC-MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3-I-mediated CaMKII inhibition. CONCLUSIONS: Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling.

Combined Na(+)/Ca(2+) exchanger and L-type calcium channel block as a potential strategy to suppress arrhythmias and maintain ventricular function.

BACKGROUND: L-type calcium channel (LTCC) and Na(+)/Ca(2+) exchanger (NCX) have been implicated in repolarization-dependent arrhythmias, but also modulate calcium and contractility. Although LTCC inhibition is negative inotropic, NCX inhibition has the opposite effect. Combined block may, therefore, offer an advantage for hemodynamics and antiarrhythmic efficiency, particularly in diseased hearts. In a model of proarrhythmia, the dog with chronic atrioventricular block, we investigated whether combined inhibition of NCX and LTCC with SEA-0400 is effective against dofetilide-induced torsade de pointes arrhythmias (TdP), while maintaining calcium homeostasis and hemodynamics. METHODS AND RESULTS: Left ventricular pressure (LVP) and ECG were monitored during infusion of SEA-0400 and verapamil in anesthetized dogs. Different doses were tested against dofetilide-induced TdP in chronic atrioventricular block dogs. In ventricular myocytes, effects of SEA-0400 were tested on action potentials, calcium transients, and early afterdepolarizations. In cardiomyocytes, SEA-0400 (1 µmol/L) blocked 66±3% of outward NCX, 50±2% of inward NCX, and 33±9% of LTCC current. SEA-0400 had no effect on systolic calcium, but slowed relaxation, despite action potential shortening, and increased diastolic calcium. SEA-0400 stabilized dofetilide-induced lability of repolarization and suppressed early afterdepolarizations. In vivo, SEA-0400 (0.4 and 0.8 mg/kg) had no effect on left ventricular pressure and suppressed dofetilide-induced TdPs dose dependently. Verapamil (0.3 mg/kg) also inhibited TdP, but caused a 15±8% drop of left ventricular pressure. A lower dose of verapamil without effects on left ventricular pressure (0.06 mg/kg) was not antiarrhythmic. CONCLUSIONS: In chronic atrioventricular block dogs, SEA-0400 treatment is effective against TdP. Unlike specific inhibition of LTCC, combined NCX and LTCC inhibition has no negative effects on cardiac hemodynamics.

Relevance of calmodulin/CaMKII activation for arrhythmogenesis in the AV block dog.

BACKGROUND: The calcium-dependent signaling molecules calcineurin and calcium/calmodulin-dependent protein kinase II (CaMKII) both have been linked to decompensated hypertrophy and arrhythmias. CaMKII is also believed to be involved in acute modulation of ion channels. OBJECTIVE: The purpose of this study was to determine the role of calcineurin and CaMKII in a dog model of compensated hypertrophy and a long QT phenotype. METHODS: AV block was created in dogs to induce ventricular remodeling, including enhanced susceptibility to dofetilide-induced torsades de pointes arrhythmias. Dogs were treated with cyclosporin A for 3 weeks, which reduced calcineurin activity, as determined by mRNA expression levels of regulator of calcineurin 1 exon 4, but which was unable to prevent structural, contractile, or electrical remodeling and arrhythmias. Biopsies were taken before and at 2 or 9 weeks after AV block. Western blots were performed against phosphorylated and total CaMKII, phospholamban, Akt, and histone deacetylase 4 (HDAC4). RESULTS: Chronic AV block showed an increase in Akt, CaMKII and phospholamban phosphorylation levels, but HDAC4 phosphorylation remained unaltered. Dofetilide induced torsades de pointes in vivo and early afterdepolarizations in cardiomyocytes, and increased [Ca(2+)](i) and CaMKII autophosphorylation. Both W-7 and KN-93 treatment counteracted this. CONCLUSION: The calcineurin pathway seems not to be involved in long-term cardiac remodeling of the chronic AV block dog. Although CaMKII is chronically activated, this does not translate to HDAC4 phosphorylation. However, acute CaMKII overactivation is able to initiate arrhythmias based on triggered activity.

Verapamil as an antiarrhythmic agent in congestive heart failure: hopping from rabbit to human?

Repolarization-dependent cardiac arrhythmias only arise in hearts facing multiple 'challenges' affecting its so-called repolarization reserve. Congestive heart failure (CHF) is one such challenge frequently observed in humans and is accompanied by altered calcium handling within the contractile heart cell. This raises the question as to whether or not the well-known calcium channel antagonist verapamil acts as an antiarrhythmic drug in this setting, as seen in arrhythmia models without CHF. According to the study of Milberg et al. in this issue of BJP, the answer is yes. The results of this study, using a rabbit CHF model, raise important questions. First, given that the model combines CHF with a number of other interventions that predispose towards arrhythmia, will similar conclusions be reached in a setting where CHF is a more prominent proarrhythmic challenge; second, what is the extent to which other effects of calcium channel block would limit the clinical viability of this pharmacological approach in CHF? In vivo studies in large animal CHF models are now required to further explore this interesting, but complex, approach to the treatment of arrhythmia. LINKED ARTICLE This article is a commentary on Milberg et al., pp. 557-568 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2011.01721.x.