Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

Comprehensive Meta-Analysis of Futile Recanalization in Acute Ischemic Stroke Patients Undergoing Endovascular Thrombectomy: Prevalence, Factors, and Clinical Outcomes.

BACKGROUND: Futile recanalization (FR) continues to raise concern despite the success of endovascular thrombectomy (EVT) in acute ischemic stroke (AIS). Understanding the prevalence of FR and identifying associated factors are crucial for refining patient prognoses and optimizing management strategies. OBJECTIVES: This study aims to comprehensively assess the pooled prevalence of FR, explore the diverse factors connected with FR, and establish the association of FR with long-term clinical outcomes among AIS patients undergoing EVT. MATERIALS AND METHODS: Incorporating studies focusing on FR following EVT in AIS patients, we conducted a random-effect meta-analysis to assess the pooled prevalence and its association with various clinical and imaging risk factors linked to FR. Summary estimates were compiled and study heterogeneity was explored. RESULTS: Our comprehensive meta-analysis, involving 11,700 AIS patients undergoing EVT, revealed a significant pooled prevalence of FR at 51%, with a range of 48% to 54% (Effect Size [ES]: 51%; 95% Confidence Interval [CI]: 48-54%; z = 47.66; p < 0.001). Numerous clinical factors demonstrated robust correlations with FR, including atrial fibrillation (Odds Ratio [OR]: 1.39, 95% CI 1.22 1.59; p < 0.001), hypertension (OR 1.65, 95% CI 1.41 1.92; p < 0.001), diabetes mellitus (OR 1.71, 95% CI 1.47 1.99; p < 0.001), previous stroke or transient ischemic attack (OR 1.298, 95% CI 1.06 1.59; p = 0.012), prior anticoagulant usage (OR 1.33, 95% CI 1.08 1.63; p = 0.007), cardioembolic strokes (OR 1.34, 95% CI 1.10 1.63; p = 0.003), and general anesthesia (OR 1.53, 95% CI 1.35 1.74; p < 0.001). Conversely, FR exhibited reduced likelihoods of smoking (OR 0.66, 95% CI 0.57 0.77; p < 0.001), good collaterals (OR 0.33, 95% CI 0.23 0.49; p < 0.001), male sex (OR 0.87, 95% CI 0.77 0.97; p = 0.016), and intravenous thrombolysis (IVT) (OR 0.75, 95% CI 0.66 0.86; p < 0.001). FR was strongly associated with increasing age (standardized mean difference [SMD] 0.49, 95% CI 0.42 0.56; p < 0.0001), baseline systolic blood pressure (SMD 0.20, 95% CI 0.13 0.27; p < 0.001), baseline National Institute of Health Stroke Severity Score (SMD 0.75, 95% CI: 0.65 0.86; p < 0.001), onset-to-treatment time (SMD 0.217, 95% CI 0.13 0.30; p < 0.001), onset-to-recanalization time (SMD 0.38, 95% CI 0.19; 0.57; p < 0.001), and baseline blood glucose (SMD 0.31, 95% CI 0.22 0.41; p < 0.001), while displaying a negative association with reduced baseline Alberta Stroke Program Early CT Score (ASPECTS) (SMD -0.37, 95% CI -0.46 -0.27; p < 0.001). Regarding clinical outcomes, FR was significantly associated with increased odds of symptomatic intracranial hemorrhages (OR 7.37, 95% CI 4.89 11.12; p < 0.001), hemorrhagic transformations (OR 2.98, 95% CI 2.37 3.75; p < 0.001), and 90-day mortality (OR 19.24, 95% CI 1.57 235.18; p = 0.021). CONCLUSIONS: The substantial prevalence of FR, standing at approximately 51%, warrants clinical consideration. These findings underscore the complexity of FR in AIS patients and highlight the importance of tailoring management strategies based on individual risk factors and clinical profiles.

Antioxidant Behavioural Phenotype in the Immp2l Gene Knock-Out Mouse.

Mitochondrial dysfunction is strongly associated with autism spectrum disorder (ASD) and the Inner mitochondrial membrane protein 2-like (IMMP2L) gene is linked to autism inheritance. However, the biological basis of this linkage is unknown notwithstanding independent reports of oxidative stress in association with both IMMP2L and ASD. To better understand IMMP2L's association with behaviour, we developed the Immp2lKD knockout (KO) mouse model which is devoid of Immp2l peptidase activity. Immp2lKD -/- KO mice do not display any of the core behavioural symptoms of ASD, albeit homozygous Immp2lKD -/- KO mice do display increased auditory stimulus-driven instrumental behaviour and increased amphetamine-induced locomotion. Due to reports of increased ROS and oxidative stress phenotypes in an earlier truncated Immp2l mouse model resulting from an intragenic deletion within Immp2l, we tested whether high doses of the synthetic mitochondrial targeted antioxidant (MitoQ) could reverse or moderate the behavioural changes in Immp2lKD -/- KO mice. To our surprise, we observed that ROS levels were not increased but significantly lowered in our new Immp2lKD -/- KO mice and that these mice had no oxidative stress-associated phenotypes and were fully fertile with no age-related ataxia or neurodegeneration as ascertained using electron microscopy. Furthermore, the antioxidant MitoQ had no effect on the increased amphetamine-induced locomotion of these mice. Together, these findings indicate that the behavioural changes in Immp2lKD -/- KO mice are associated with an antioxidant-like phenotype with lowered and not increased levels of ROS and no oxidative stress-related phenotypes. This suggested that treatments with antioxidants are unlikely to be effective in treating behaviours directly resulting from the loss of Immp2l/IMMP2L activity, while any behavioural deficits that maybe associated with IMMP2L intragenic deletion-associated truncations have yet to be determined.

HISTOLOGY, DIMENSIONS, AND FLUORESCEIN STAINING CHARACTERISTICS OF NODULAR AND CUTICULAR DRUSEN IN AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To enable in vivo analysis of drusen composition and lifecycle, the macular nodular and cuticular drusen were assessed using histology. METHODS: Median and interquartile range of base widths of single (nonconfluent) nodular drusen in three sources were determined histologically: 43 eyes of 43 clinically undocumented donors, in an online resource; one eye with punctate hyperfluorescence in fluorescein angiography; and two eyes of one patient with bilateral "starry sky" cuticular drusen. All tissues were processed for high-resolution epoxy-resin histology and for cuticular drusen, transmission electron microscopy. RESULTS: All drusen localized between the retinal pigment epithelium basal lamina and inner collagenous layer of the Bruch membrane. They were solid, globular, homogeneously stained with toluidine blue, and uncovered by basal laminar deposit and basal mounds. Median base widths were 13.0 µ m (Source 1, N = 128 drusen, interquartile range 7.7, 20.0 µ m), 15.3 µ m (Source 2, N = 87, interquartile range 10.6, 20.5 µ m), and 7.3 µ m (Source 3, N = 78, interquartile range 3.9, 14.1 µ m). CONCLUSION: In three samples, >90% of solitary nodular drusen were <30 µ m, the visibility threshold in color fundus photography; these drusen are hyperfluorescent in fluorescein angiography. Whether these progress to soft drusen, known as high-risk from epidemiology studies and hypofluorescent, may be determinable from multimodal imaging datasets that include fluorescein angiography.

Prognostic accuracy and impact of cerebral collateral status on clinical and safety outcomes in acute ischemic stroke patients receiving reperfusion therapy: a systematic meta-analysis.

BACKGROUND: Cerebral collateral status has a potential role in mediating postreperfusion clinical and safety outcomes in acute ischemic stroke (AIS). PURPOSE: To investigate the prognostic accuracy and impact of collateral status on clinical and safety outcomes in patients with AIS receiving reperfusion therapy. MATERIAL AND METHODS: Studies with AIS patients treated with reperfusion therapy, collateral status assessed using Tan, ASITN/SIR, or similar collateral grading methods and data stratified according to collateral status were included. Relevant data on clinical outcomes, such as functional outcome at 90 days, mortality at 90 days, angiographic reperfusion, symptomatic intracerebral hemorrhage (sICH) and hemorrhagic transformation (HT), were collated and analyzed. RESULTS: A meta-analysis of 18 studies involving 4132 patients with AIS was conducted. Good collateral status was significantly associated with angiographic reperfusion (odds ratio [OR]=1.97, 95% confidence interval [CI]=1.38-2.80; P < 0.0001), sICH (OR=0.67, 95% CI=0.46-0.99; P = 0.042), and 90-day functional outcome (OR=3.05, 95% CI=1.78-5.24; P < 0.0001). However, its association with HT (OR=0.76, 95% CI=0.38-1.51; P = 0.425) and three-month mortality (OR=0.53, 95% CI=0.17-1.69; P = 0.280) did not reach statistical significance. The prognostic accuracy of collaterals for predicting angiographic reperfusion, HT, functional outcome (at 90 days), and mortality (at 90 days) were 63%, 49%, 66%, and 48%, respectively. CONCLUSION: Cerebral collaterals are significantly associated with clinical and safety outcomes, albeit with a prognostic accuracy range of 48%-66%; thus, evaluation of their patency is a useful prognostic tool in patients with AIS receiving reperfusion therapy.

Association of Lesion Topography with Functional Outcomes in Acute Ischemic Stroke Patients Considered for, or Receiving, Reperfusion Therapy: A Meta-Analysis.

Background: The impact of lesion topography (LT), characterised by the Alberta Stroke Programme Early CT Score (ASPECTS), on outcomes after reperfusion therapy in acute ischemic stroke (AIS) is poorly elucidated. We investigated the prognostic accuracy of ASPECTS-based LT assessment and its association with clinical outcomes in AIS patients considered for reperfusion therapy or receiving intravenous thrombolysis (IVT), endovascular thrombectomy (EVT), or none or both. Methods: Studies were identified from PubMed with additional studies added from Google Scholar. The prevalence of individual ASPECTS regions will also be determined. The association of individual ASPECTS regions with the functional outcome at 90 days will be assessed using random-effects modelling for various cut-offs, such as 6, 7 and 8. The association of continuous ASPECTS with the functional outcome at 90 days will also be undertaken. Forest plots of odds ratios (ORs) will be generated. Results: A total of 25 studies have been included in the final analysis, encompassing 11,404 patients. Pooled estimates indicate that the highest prevalence rates were in cases involving the insula and lentiform nucleus. Subgroup analysis for ASPECTS < 6 (OR 6.10; 95% CI 2.50−14.90; p < 0.0001), ASPECTS < 7 (OR 4.58; 95% CI 1.18−17.86; p < 0.0001) and ASPECTS < 8 (OR 2.26; 95% CI 1.32−3.89; p < 0.0001) revealed a significant association with poor functional outcome at 90 days. Decreasing ASPECTS significantly increased the odds of poor functional outcomes at 90 days (SMD −1.15; 95% CI −1.77−−0.52; p < 0.0001). Conclusions: Our meta-analysis demonstrates that decreasing ASPECTS is significantly associated with poor functional outcomes. Individual ASPECTS regions associated with the highest odds of poor functional outcomes were identified. Future studies on the association of LT and clinical outcomes specific to EVT are required.

Is Composition of Brain Clot Retrieved by Mechanical Thrombectomy Associated with Stroke Aetiology and Clinical Outcomes in Acute Ischemic Stroke?-A Systematic Review and Meta-Analysis.

Background: Brain clots retrieved following endovascular thrombectomy in acute ischemic stroke patients may offer unique opportunities to characterise stroke aetiology and aid stroke decision-making in select groups of patients. However, the evidence around the putative association of clot morphology with stroke aetiology is limited and remains inconclusive. This study aims to perform a systematic review and meta-analysis to delineate the association of brain clot composition with stroke aetiology and post-reperfusion outcomes in patients receiving endovascular thrombectomy. Methods: The authors conducted a systematic literature review and meta-analysis by extracting data from several research databases (MEDLINE/PubMed, Cochrane, and Google Scholar) published since 2010. We used appropriate key search terms to identify clinical studies concerning stroke thrombus composition, aetiology, and clinical outcomes, in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Results: The authors identified 30 articles reporting on the relationship between stroke thrombus composition or morphology and aetiology, imaging, or clinical outcomes, of which 21 were included in the meta-analysis. The study found that strokes of cardioembolic origin (SMD = 0.388; 95% CI, 0.032-0.745) and cryptogenic origin (SMD = 0.468; 95% CI, 0.172-0.765) had significantly higher fibrin content than strokes of non-cardioembolic origin. Large artery atherosclerosis strokes had significantly lower fibrin content than cardioembolic (SMD = 0.552; 95% CI, 0.099-1.004) or cryptogenic (SMD = 0.455; 95% CI, 0.137-0.774) strokes. Greater red blood cell content was also significantly associated with a thrombolysis in cerebral infarction score of 2b-3 (SMD = 0.450; 95% CI, 0.177-0.722), and a positive hyperdense middle cerebral artery sign (SMD = 0.827; 95% CI, 0.472-1.183). No significant associations were found between red blood cell, platelet, or white blood cell content and aetiology, or between clot composition and bridging thrombolysis. Conclusions: This meta-analysis found that fibrin composition is significantly higher in strokes of cardioembolic and cryptogenic origin, and that red blood cell content is positively associated with the hyperdense middle cerebral artery sign and better reperfusion outcomes. Important advances to stroke clinical workup can be derived from these findings, in which many aspects of stroke workflow remain to be optimised. As data are still limited in terms of the association of various thrombus components with stroke aetiology as well as a standardised method of analysis, further studies are required to validate these findings to guide their use in clinical decision-making.

Digital subtraction angiography in cerebrovascular disease: current practice and perspectives on diagnosis, acute treatment and prognosis.

Digital Subtraction Angiography (DSA) is the gold-standard imaging modality in acute cerebrovascular diagnosis. The role of DSA has become increasingly prominent since the incorporation of endovascular therapy in standards of care for acute ischemic stroke. It is used in the assessment of cerebral vessel patency; however, the therapeutic role of DSA from a prognostic standpoint merits further investigation. The current paper provides an update on current practice on diagnostic, therapeutic and prognostic use of DSA in acute cerebrovascular diseases and various indications and perspectives that may apply, or limit its use, in ongoing surveillance or prognosis. Pre-clinical and clinical studies on the aspects, including but not limited to the morphology of cerebrovasculature in acute ischaemic stroke, are required to delineate and inform its prognostic role.

Role of diabetes in stroke: Recent advances in pathophysiology and clinical management.

The increasing prevalence of diabetes and stroke is a major global public health concern. Specifically, acute stroke patients, with pre-existing diabetes, pose a clinical challenge. It is established that diabetes is associated with a worse prognosis after acute stroke and the various biological factors that mediate poor recovery profiles in diabetic patients is unknown. The level of association and impact of diabetes, in the setting of reperfusion therapy, is yet to be determined. This article presents a comprehensive overview of the current knowledge of the role of diabetes in stroke, therapeutic strategies for primary and secondary prevention of cardiovascular disease and/or stroke in diabetes, and various therapeutic considerations that may apply during pre-stroke, acute, sub-acute and post-stroke stages. The early diagnosis of diabetes as a comorbidity for stroke, as well as tailored post-stroke management of diabetes, is pivotal to our efforts to limit the burden. Increasing awareness and involvement of neurologists in the management of diabetes and other cardiovascular risk factors is desirable towards improving stroke prevention and efficacy of reperfusion therapy in acute stroke patients with diabetes.

Prognostic capacity of hyperdense middle cerebral artery sign in anterior circulation acute ischaemic stroke patients receiving reperfusion therapy: a systematic review and meta-analysis.

Pre-intervention CT imaging-based biomarkers, such as hyperdense middle cerebral artery sign (HMCAS) may have a role in acute ischaemic stroke prognostication. However, the clinical utility of HMCAS in settings of reperfusion therapy and the level of prognostic association is still unclear. This systematic review and meta-analysis investigated the association of HMCAS sign with clinical outcomes and its prognostic capacity in acute ischaemic stroke patients treated with reperfusion therapy. Prospective and retrospective studies from the following databases were retrieved from EMBASE, MEDLINE and Cochrane. Association of HMCAS with functional outcome, symptomatic intracerebral haemorrhage (sICH) and mortality were investigated. The random effect model was used to calculate the risk ratio (RR). Subgroup analyses were performed for subgroups of patients receiving thrombolysis (tPA), mechanical thrombectomy (EVT) and/or combined therapy (tPA + EVT). HMCAS significantly increased the rate of poor functional outcome by 1.43-fold in patients (RR 1.43; 95% CI 1.30-1.57; p < 0.0001) without any significant differences in sICH rates (RR 0.91; 95% CI 0.68-1.23; p = 0.546) and mortality (RR 1.34; 95% CI 0.72-2.51; p = 354) in patients with positive HMCAS as compared to negative HMCAS. In subgroup analyses, significant association between HMCAS and 90 days functional outcome was observed in patients receiving tPA (RR 1.53; 95% CI 1.40-1.67; p < 0.0001) or both therapies (RR 1.40; 95% CI 1.08-1.80; p = 0.010). This meta-analysis demonstrated that pre-treatment HMCAS increases risk of poor functional outcomes. However, its prognostic sensitivity and specificity in predicting long-term functional outcome, mortality and sICH after reperfusion therapy is poor.

Stroke Aetiology and Collateral Status in Acute Ischemic Stroke Patients Receiving Reperfusion Therapy-A Meta-Analysis.

BACKGROUND: The interplay between collateral status and stroke aetiology may be crucial in the evaluation and management of acute ischemic stroke (AIS). Our understanding of this relationship and its level of association remains sub-optimal. This study sought to examine the association of pre-intervention collateral status with stroke aetiology, specifically large artery atherosclerosis (LAA) and cardio-embolism (CE), in AIS patients receiving reperfusion therapy, by performing a meta-analysis. METHODS: Relevant search terms were explored on Medline/PubMed, Embase and Cochrane databases. Studies were included using the following inclusion criteria: (a) patients aged 18 or above; (b) AIS patients; (c) patients receiving reperfusion therapy; (d) total cohort size of >20, and (e) qualitative or quantitative assessment of pre-intervention collateral status on imaging using a grading scale. Random-effects meta-analysis was performed to investigate the association of aetiology with pre-intervention collateral status, and forest plots of risk ratio (RR) were generated. RESULTS: A meta-analysis was conducted on seven studies, with a cumulative cohort of 1235 patients, to assess the association of pre-intervention collateral status with stroke aetiology. Patients with LAA were associated significantly with an increased rate of good collaterals (RR 1.24; 95% CI 1.04-1.50; p = 0.020, z = 2.33). Contrarily, CE aetiology was associated significantly with a decreased rate of good collaterals (RR 0.83; 95% CI 0.71-0.98; p = 0.027, z = -2.213). CONCLUSIONS: This study demonstrates that, in AIS patients receiving reperfusion therapy, LAA and CE aetiologies are associated significantly with collateral status.

Diagnostic and prognostic utility of computed tomography perfusion imaging in posterior circulation acute ischemic stroke: A systematic review and meta-analysis.

BACKGROUND: Computed tomography perfusion (CTP) imaging could be useful in the diagnosis of posterior circulation stroke (PCS) and in identifying patients who are likely to experience favorable outcomes following reperfusion therapy. The current study sought to investigate the diagnostic and prognostic capability of CTP in acute ischemic PCS by performing a systematic review and meta-analysis. METHODS: Medline/PubMed and the Cochrane Library were searched using the terms: "posterior circulation", "CT perfusion", "acute stroke", and "reperfusion therapy". The following studies were included: (1) patients aged 18 years or above; (2) patients diagnosed with PCS; and (3) studies with good methodological design. Pooled sensitivity (SENS), specificity (SPEC), and area under the curve (AUC), computed using the summary receiver operating characteristic (SROC) curves, were used to determine diagnostic/prognostic capability. RESULTS: Out of 14 studies included, a meta-analysis investigating diagnostic accuracy of CTP was performed on nine studies. Meta-analysis demonstrated comparable diagnostic accuracy of CTP to non-contrast computed tomography (NCCT) (AUCCTP : 0.90 [95% CI 0.87-0.92] vs. AUCNCCT : 0.96 [95% CI 0.94-0.97]); however, with higher pooled sensitivity (SENSCTP : 72% [95% CI 57%-83%] vs. SENSNCCT : 25% [95% CI 17%-35%]) and lower specificity (SPECCTP : 90% [95% CI 83%-94%] vs. SPECNCCT : 96% [95% CI 95%-98%]) than NCCT. Meta-analysis to determine prognostic capability of CTP could not be performed. CONCLUSIONS: CTP has limited diagnostic utility in acute ischemic PCS, albeit with superior diagnostic sensitivity and inferior diagnostic specificity to NCCT. Further prospective trials are required to validate the prognostic capability of CTP-derived parameters in PCS.

Cerebral collaterals in acute ischaemia: Implications for acute ischaemic stroke patients receiving reperfusion therapy.

The cerebral collaterals play an important role in penumbral tissue sustenance after an acute ischaemic stroke. Recent studies have demonstrated the potential role of collaterals in the selection of acute ischaemic stroke patients eligible for reperfusion therapy. However, the understanding of the significance and evidence around the role of collateral status in predicting outcomes in acute ischaemic stroke patients treated with reperfusion therapy is still unclear. Moreover, the use of pre-treatment collaterals in patient selection and prognosis is relatively underappreciated in clinical settings. A focused review of the literature was performed on the various methods of collateral evaluation and the role of collateral status in acute ischaemic stroke patients receiving reperfusion therapy. We discuss the methods of evaluating pre-treatment collaterals in clinical settings. The patient selection based on collateral status as well as the prognostic and therapeutic value of collaterals in acute ischaemic stroke, in settings of intravenous thrombolysis or endovascular therapy alone, and bridge therapy, are summarized. Recommendations for future research and possible pharmacological intervention strategies aimed at collateral enhancement are also discussed. Collaterals may play an important role in identifying acute ischaemic stroke patients who are likely to benefit from endovascular treatment in an extended time window. Future neuroscientific efforts to better improve our understanding of the role of collaterals in acute ischaemia as well as clinical studies to delineate its role in patient selection and acute stroke prognosis are warranted.

A simplified method for producing human lens epithelial cells and light-focusing micro-lenses from pluripotent stem cells.

Here we describe a modified method for harvesting tens-of-millions of human lens epithelial-like cells from differentiated pluripotent stem cell cultures. To assess the utility of this method, we analysed the lens cell population via: light microscopy; single cell RNA-sequencing and gene ontology analyses; formation of light-focusing micro-lenses; mass spectrometry; and electron microscopy. Both individually and collectively, the data indicate this simplified harvesting method provides a large-scale source of stem cell-derived lens cells and micro-lenses for investigating human lens and cataract formation.

Scanning electron microscopy as a new tool for diagnostic pathology and cell biology.

Scanning electron microscopy (SEM) use in the biomedical sciences has traditionally been used for characterisation of cell and tissue surface topography. This paper demonstrates the utility of high-resolution scanning electron microscopy (HRSEM) to diagnostic pathology and cell biology ultrastructural examinations. New SEM applications based on the production of transmission electron microscopy-like (TEM-like) images are now possible with the recent introduction of new technologies such as low kV scanning transmission electron microscopy (STEM) detectors, automated scan generators and high-resolution column configurations capable of sub-nanometre resolution. Typical specimen types traditionally imaged by TEM have been examined including renal, lung, prostate and brain tissues. The specimen preparation workflow was unchanged from that routinely used to prepare TEM tissue, apart from replacing copper grids for section mounting with a silicon substrate. These instruments feature a small footprint with little in the way of ancillary equipment, such as water chillers, and are more cost-effective than traditional TEM columns. Also, a new generation of benchtop SEMs have recently become available and have also been assessed for its utility in the tissue pathology and cell biology settings.

Clot Histopathology in Ischemic Stroke with Infective Endocarditis.

BACKGROUND: Endovascular thrombectomy (EVT) has shown efficacy in acute ischemic stroke (AIS) patients with infective endocarditis (IE). The possibility to undertake advanced histopathological clot analysis following EVT offers a new avenue to establish the etiological basis of the stroke - which is often labelled "cryptogenic." In this paper, we present our findings from four consecutive patients with IE who underwent EVT following an AIS at our tertiary referral comprehensive stroke centre. METHODS: Comprehensive histopathological analysis of clot retrieved after EVT, including morphology, was undertaken. RESULTS: The consistent observation was the presence of dense paucicellular fibrinoid material mixed/interspersed with clusters of bacterial cocci. This clot morphology may be specific to septic embolus due to IE unlike incidental bacteraemia and could possibly explain the refractoriness of such clots to systemic thrombolysis. CONCLUSION: Detailed morphological and histopathological analysis of EVT-retrieved clots including Gram staining can assist in etiological classification of the clot. Understanding the composition of the clot may be of clinical value in early diagnostics and mapping treatment planning in IE.

Differences in LC3B expression and prognostic implications in oropharyngeal and oral cavity squamous cell carcinoma patients.

BACKGROUND: This study examined the prognostic significance of microtubule-associated protein light chain 3B (LC3B) expression in oropharyngeal and oral cavity squamous cell carcinoma (SCC). The prognostic significance of LC3B expression in relation to human papillomavirus (HPV) status in oropharyngeal SCC was also examined. METHODS: Tissue microarrays (TMAs) were constructed from formalin-fixed, paraffin-embedded oropharyngeal (n = 47) and oral cavity (n = 95) SCC tissue blocks from patients with long-term recurrence and overall survival data (median = 47 months). LC3B expression on tumour was assessed by immunohistochemistry and evaluated for associations with clinicopathological variables. LC3B expression was stratified into high and low expression cohorts using ROC curves with Manhattan distance minimisation, followed by Kaplan-Meier and multivariable survival analyses. Interaction terms between HPV status and LC3B expression in oropharyngeal SCC patients were also examined by joint-effects and stratified analyses. RESULTS: Kaplan-Meier survival and univariate analyses revealed that high LC3B expression was correlated with poor overall survival in oropharyngeal SCC patients (p = 0.007 and HR = 3.18, 95% CI 1.31-7.71, p = 0.01 respectively). High LC3B expression was also an independent prognostic factor for poor overall survival in oropharyngeal SCC patients (HR = 4.02, 95% CI 1.38-11.47, p = 0.011). In contrast, in oral cavity SCC, only disease-free survival remained statistically significant after univariate analysis (HR = 2.36, 95% CI 1.19-4.67, p = 0.014), although Kaplan-Meier survival analysis showed that high LC3B expression correlated with poor overall and disease-free survival (p = 0.046 and 0.011 respectively). Furthermore, oropharyngeal SCC patients with HPV-negative/high LC3B expression were correlated with poor overall survival in both joint-effects and stratified presentations (p = 0.024 and 0.032 respectively). CONCLUSIONS: High LC3B expression correlates with poor prognosis in oropharyngeal and oral cavity SCC, which highlights the importance of autophagy in these malignancies. High LC3B expression appears to be an independent prognostic marker for oropharyngeal SCC but not for oral cavity SCC patients. The difference in the prognostic significance of LC3B between oropharyngeal and oral cavity SCCs further supports the biological differences between these malignancies. The possibility that oropharyngeal SCC patients with negative HPV status and high LC3B expression were at particular risk of a poor outcome warrants further investigation in prospective studies with larger numbers.

Cuticular Drusen: Clinical Phenotypes and Natural History Defined Using Multimodal Imaging.

PURPOSE: To define the range and life cycles of cuticular drusen phenotypes using multimodal imaging and to review the histologic characteristics of cuticular drusen. DESIGN: Retrospective, observational cohort study and experimental laboratory study. PARTICIPANTS: Two hundred forty eyes of 120 clinic patients with a cuticular drusen phenotype and 4 human donor eyes with cuticular drusen (n = 2), soft drusen (n = 1), and hard drusen (n = 1). METHODS: We performed a retrospective review of clinical and multimodal imaging data of patients with a cuticular drusen phenotype. Patients had undergone imaging with various combinations of color photography, fluorescein angiography, indocyanine green angiography, near-infrared reflectance, fundus autofluorescence, high-resolution OCT, and ultrawide-field imaging. Human donor eyes underwent processing for high-resolution light and electron microscopy. MAIN OUTCOME MEASURES: Appearance of cuticular drusen in multimodal imaging and the topography of a cuticular drusen distribution; age-dependent variations in cuticular drusen phenotypes, including the occurrence of retinal pigment epithelium (RPE) abnormalities, choroidal neovascularization, acquired vitelliform lesions (AVLs), and geographic atrophy (GA); and ultrastructural and staining characteristics of druse subtypes. RESULTS: The mean age of patients at the first visit was 57.9±13.4 years. Drusen and RPE changes were seen in the peripheral retina, anterior to the vortex veins, in 21.8% of eyes. Of eyes with more than 5 years of follow-up, cuticular drusen disappeared from view in 58.3% of eyes, drusen coalescence was seen in 70.8% of eyes, and new RPE pigmentary changes developed in 56.2% of eyes. Retinal pigment epithelium abnormalities, AVLs, neovascularization, and GA occurred at a frequency of 47.5%, 24.2%, 12.5%, and 25%, respectively, and were significantly more common in patients older than 60 years of age (all P < 0.015). Occurrence of GA and neovascularization were important determinants of final visual acuity in eyes with the cuticular drusen phenotype (both P < 0.015). Small cuticular drusen typically demonstrated a homogenous ultrastructural appearance similar to hard drusen, whereas fragmentation of the central and basal contents was seen frequently in larger cuticular drusen. CONCLUSIONS: Although the ultrastructural characteristics of cuticular drusen appear more similar to those of hard drusen, their lifecycle and macular complications are more comparable with those of soft drusen. Cuticular drusen phenotype may confer a unique risk for the development of GA and neovascularization.

Light-focusing human micro-lenses generated from pluripotent stem cells model lens development and drug-induced cataract in vitro.

Cataracts cause vision loss and blindness by impairing the ability of the ocular lens to focus light onto the retina. Various cataract risk factors have been identified, including drug treatments, age, smoking and diabetes. However, the molecular events responsible for these different forms of cataract are ill-defined, and the advent of modern cataract surgery in the 1960s virtually eliminated access to human lenses for research. Here, we demonstrate large-scale production of light-focusing human micro-lenses from spheroidal masses of human lens epithelial cells purified from differentiating pluripotent stem cells. The purified lens cells and micro-lenses display similar morphology, cellular arrangement, mRNA expression and protein expression to human lens cells and lenses. Exposing the micro-lenses to the emergent cystic fibrosis drug Vx-770 reduces micro-lens transparency and focusing ability. These human micro-lenses provide a powerful and large-scale platform for defining molecular disease mechanisms caused by cataract risk factors, for anti-cataract drug screening and for clinically relevant toxicity assays.