Association of sildenafil use with age-related macular degeneration: a retrospective cohort study.

OBJECTIVE: Despite significant advances in clinical care and understanding of the underlying pathophysiology, age-related macular degeneration (AMD)-a major cause of global blindness-lacks effective treatment to prevent the irreversible degeneration of photoreceptors leading to central vision loss. Limited studies suggest phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, may prevent AMD by increasing retinal blood flow. This study explores the potential association between sildenafil use and AMD risk in men with erectile dysfunction using UK data. METHODS AND ANALYSIS: Using the UK's IQVIA Medical Research Data, the study analysed 31 575 men prescribed sildenafil for erectile dysfunction and no AMD history from 2007 to 2015, matched with a comparator group of 62 155 non-sildenafil users in a 1:2 ratio, over a median follow-up of approximately three years. RESULTS: The primary outcome was the incidence of AMD in the two groups. The study found no significant difference in AMD incidence between the sildenafil users and the non-users, with an adjusted hazard ratio (HR) of 0.99 (95% CI 0.84 to 1.16), after accounting for confounders such as age, ethnicity, Townsend deprivation quintile, body mass index category, and diagnosis of hypertension and type 2 diabetes. CONCLUSION: The study results indicated no significant association between sildenafil use and AMD prevention in UK men with erectile dysfunction, suggesting sildenafil's protective effect on AMD is likely insignificant.

Unraveling the impact of cancer-associated fibroblasts on hypovascular pancreatic neuroendocrine tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) with low microvessel density and fibrosis often exhibit clinical aggressiveness. Given the contribution of cancer-associated fibroblasts (CAFs) to the hypovascular fibrotic stroma in pancreatic ductal adenocarcinoma, investigating whether CAFs play a similar role in PNETs becomes imperative. In this study, we investigated the involvement of CAFs in PNETs and their effects on clinical outcomes. METHODS: We examined 79 clinical PNET specimens to evaluate the number and spatial distribution of α-smooth muscle actin (SMA)-positive cells, which are indicative of CAFs. Then, the findings were correlated with clinical outcomes. In vitro and in vivo experiments were conducted to assess the effects of CAFs (isolated from clinical specimens) on PNET metastasis and growth. Additionally, the role of the stromal-cell-derived factor 1 (SDF1)-AGR2 axis in mediating communication between CAFs and PNET cells was investigated. RESULTS: αSMA-positive and platelet-derived growth factor-α-positive CAFs were detected in the hypovascular stroma of PNET specimens. A higher abundance of α-SMA-positive CAFs within the PNET stroma was significantly associated with a higher level of clinical aggressiveness. Notably, conditioned medium from PNET cells induced an inflammatory phenotype in isolated CAFs. These CAFs promoted PNET growth and metastasis. Mechanistically, PNET cells secreted interleukin-1, which induced the secretion of SDF1 from CAFs. This cascade subsequently elevated AGR2 expression in PNETs, thereby promoting tumor growth and metastasis. The downregulation of AGR2 in PNET cells effectively suppressed the CAF-mediated promotion of PNET growth and metastasis. CONCLUSION: CAFs drive the growth and metastasis of aggressive PNETs. The CXCR4-SDF1 axis may be a target for antistromal therapy in the treatment of PNET. This study clarifies mechanisms underlying PNET aggressiveness and may guide future therapeutic interventions targeting the tumor microenvironment.

Oncogenic KRAS, Mucin 4, and Activin A-Mediated Fibroblast Activation Cooperate for PanIN Initiation.

Over 90% of patients with pancreatic ductal adenocarcinoma (PDAC) have oncogenic KRAS mutations. Nevertheless, mutated KRAS alone is insufficient to initiate pancreatic intraepithelial neoplasia (PanIN), the precursor of PDAC. The identities of the other factors/events required to drive PanIN formation remain elusive. Here, optic-clear 3D histology is used to analyze entire pancreases of 2-week-old Pdx1-Cre; LSL-KrasG12D/+ (KC) mice to detect the earliest emergence of PanIN and observed that the occurrence is independent of physical location. Instead, it is found that the earliest PanINs overexpress Muc4 and associate with αSMA+ fibroblasts in both transgenic mice and human specimens. Mechanistically, KrasG12D/+ pancreatic cells upregulate Muc4 through genetic alterations to increase proliferation and fibroblast recruitments via Activin A secretion and consequently enhance cell transformation for PanIN formation. Inhibition of Activin A signaling using Follistatin (FST) diminishes early PanIN-associated fibroblast recruitment, effectively curtailing PanIN initiation and growth in KC mice. These findings emphasize the vital role of interactions between oncogenic KrasG12D/+ -driven genetic alterations and induced microenvironmental changes in PanIN initiation, suggesting potential avenues for early PDAC diagnostic and management approaches.

A Datasheet for the INSIGHT Birmingham, Solihull, and Black Country Diabetic Retinopathy Screening Dataset.

Purpose: Diabetic retinopathy (DR) is the most common microvascular complication associated with diabetes mellitus (DM), affecting approximately 40% of this patient population. Early detection of DR is vital to ensure monitoring of disease progression and prompt sight saving treatments as required. This article describes the data contained within the INSIGHT Birmingham, Solihull, and Black Country Diabetic Retinopathy Dataset. Design: Dataset descriptor for routinely collected eye screening data. Participants: All diabetic patients aged 12 years and older, attending annual digital retinal photography-based screening within the Birmingham, Solihull, and Black Country Eye Screening Programme. Methods: The INSIGHT Health Data Research Hub for Eye Health is a National Health Service (NHS)-led ophthalmic bioresource that provides researchers with safe access to anonymized, routinely collected data from contributing NHS hospitals to advance research for patient benefit. This report describes the INSIGHT Birmingham, Solihull, and Black Country DR Screening Dataset, a dataset of anonymized images and linked screening data derived from the United Kingdom's largest regional DR screening program. Main Outcome Measures: This dataset consists of routinely collected data from the eye screening program. The data primarily include retinal photographs with the associated DR grading data. Additional data such as corresponding demographic details, information regarding patients' diabetic status, and visual acuity data are also available. Further details regarding available data points are available in the supplementary information, in addition to the INSIGHT webpage included below. Results: At the time point of this analysis (December 31, 2019), the dataset comprised 6 202 161 images from 246 180 patients, with a dataset inception date of January 1, 2007. The dataset includes 1 360 547 grading episodes between R0M0 and R3M1. Conclusions: This dataset descriptor article summarizes the content of the dataset, how it has been curated, and what its potential uses are. Data are available through a structured application process for research studies that support discovery, clinical evidence analyses, and innovation in artificial intelligence technologies for patient benefit. Further information regarding the data repository and contact details can be found at https://www.insight.hdrhub.org/. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Metformin and risk of age-related macular degeneration in individuals with type 2 diabetes: a retrospective cohort study.

BACKGROUND: Age-related macular degeneration (AMD) in its late stages is a leading cause of sight loss in developed countries. Some previous studies have suggested that metformin may be associated with a reduced risk of developing AMD, but the evidence is inconclusive. AIMS: To explore the relationship between metformin use and development of AMD among patients with type 2 diabetes in the UK. METHODS: A large, population-based retrospective open cohort study with a time-dependent exposure design was carried out using IQVIA Medical Research Data, 1995-2019. Patients aged ≥40 with diagnosed type 2 diabetes were included.The exposed group was those prescribed metformin (with or without any other antidiabetic medications); the comparator (unexposed) group was those prescribed other antidiabetic medications only. The exposure status was treated as time varying, collected at 3-monthly time intervals.Extended Cox proportional hazards regression was used to calculate the adjusted HRs for development of the outcome, newly diagnosed AMD. RESULTS: A total of 173 689 patients, 57% men, mean (SD) age 62.8 (11.6) years, with incident type 2 diabetes and a record of one or more antidiabetic medications were included in the study. Median follow-up was 4.8 (IQR 2.3-8.3, range 0.5-23.8) years. 3111 (1.8%) patients developed AMD. The adjusted HR for diagnosis of AMD was 1.02 (95% CI 0.92 to 1.12) in patients prescribed metformin (with or without other antidiabetic medications) compared with those prescribed any other antidiabetic medication only. CONCLUSION: We found no evidence that metformin was associated with risk of AMD in primary care patients requiring treatment for type 2 diabetes.

Predicting the immediate impact of national lockdown on neovascular age-related macular degeneration and associated visual morbidity: an INSIGHT Health Data Research Hub for Eye Health report.

OBJECTIVE: Predicting the impact of neovascular age-related macular degeneration (nAMD) service disruption on visual outcomes following national lockdown in the UK to contain SARS-CoV-2. METHODS AND ANALYSIS: This retrospective cohort study includes deidentified data from 2229 UK patients from the INSIGHT Health Data Research digital hub. We forecasted the number of treatment-naïve nAMD patients requiring anti-vascular endothelial growth factor (anti-VEGF) initiation during UK lockdown (16 March 2020 through 31 July 2020) at Moorfields Eye Hospital (MEH) and University Hospitals Birmingham (UHB). Best-measured visual acuity (VA) changes without anti-VEGF therapy were predicted using post hoc analysis of Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD trial sham-control arm data (n=238). RESULTS: At our centres, 376 patients were predicted to require anti-VEGF initiation during lockdown (MEH: 325; UHB: 51). Without treatment, mean VA was projected to decline after 12 months. The proportion of eyes in the MEH cohort predicted to maintain the key positive visual outcome of ≥70 ETDRS letters (Snellen equivalent 6/12) fell from 25.5% at baseline to 5.8% at 12 months (UHB: 9.8%-7.8%). Similarly, eyes with VA <25 ETDRS letters (6/96) were predicted to increase from 4.3% to 14.2% at MEH (UHB: 5.9%-7.8%) after 12 months without treatment. CONCLUSIONS: Here, we demonstrate how combining data from a recently founded national digital health data repository with historical industry-funded clinical trial data can enhance predictive modelling in nAMD. The demonstrated detrimental effects of prolonged treatment delay should incentivise healthcare providers to support nAMD patients accessing care in safe environments. TRIAL REGISTRATION NUMBER: NCT00056836.

Structural basis of interleukin-17B receptor in complex with a neutralizing antibody for guiding humanization and affinity maturation.

Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.

Interplays of glucose metabolism and KRAS mutation in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancer worldwide. The primary reasons for this are the lack of early detection methods and targeted therapy. Emerging evidence highlights the metabolic addiction of cancer cells as a potential target to combat PDAC. Oncogenic mutations of KRAS are the most common triggers that drive glucose uptake and utilization via metabolic reprogramming to support PDAC growth. Conversely, high glucose levels in the pancreatic microenvironment trigger genome instability and de novo mutations, including KRASG12D, in pancreatic cells through metabolic reprogramming. Here, we review convergent and diverse metabolic networks related to oncogenic KRAS mutations between PDAC initiation and progression, emphasizing the interplay among oncogenic mutations, glucose metabolic reprogramming, and the tumor microenvironment. Recognizing cancer-related glucose metabolism will provide a better strategy to prevent and treat the high risk PDAC population.

Building trust in real-world data: lessons from INSIGHT, the UK's health data research hub for eye health and oculomics.

PURPOSE OF REVIEW: In this review, we consider the challenges of creating a trusted resource for real-world data in ophthalmology, based on our experience of establishing INSIGHT, the UK's Health Data Research Hub for Eye Health and Oculomics. RECENT FINDINGS: The INSIGHT Health Data Research Hub maximizes the benefits and impact of historical, patient-level UK National Health Service (NHS) electronic health record data, including images, through making it research-ready including curation and anonymisation. It is built around a shared 'north star' of enabling research for patient benefit. INSIGHT has worked to establish patient and public trust in the concept and delivery of INSIGHT, with efficient and robust governance processes that support safe and secure access to data for researchers. By linking to systemic data, there is an opportunity for discovery of novel ophthalmic biomarkers of systemic diseases ('oculomics'). Datasets that provide a representation of the whole population are an important tool to address the increasingly recognized threat of health data poverty. SUMMARY: Enabling efficient, safe access to routinely collected clinical data is a substantial undertaking, especially when this includes imaging modalities, but provides an exceptional resource for research. Research and innovation built on inclusive real-world data is an important tool in ensuring that discoveries and technologies of the future may not only favour selected groups, but also work for all patients.

Differential effects of glucose and N-acetylglucosamine on genome instability.

Aberrant sugar metabolism is linked to an increased risk of pancreatic cancer. Previously, we found that high glucose induces genome instability and de novo oncogenic KRAS mutation preferentially in pancreatic cells through dysregulation of O-GlcNAcylation. Increasing O-GlcNAcylation by extrinsically supplying N-acetyl-D-glucosamine (GlcNAc) causes genome instability in all kinds of cell types regardless of pancreatic origin. Since many people consume excessive amount of sugar (glucose, fructose, and sucrose) in daily life, whether high sugar consumption directly causes genome instability in animals remains to be elucidated. In this communication, we show that excess sugar in the daily drink increases DNA damage and protein O-GlcNAcylation preferentially in pancreatic tissue but not in other kinds of tissue of mice. The effect of high sugar on the pancreatic tissue may be attributed to the intrinsic ratio of GFAT and PFK activity, a limiting factor that dictates UDP-GlcNAc levels. On the other hand, GlcNAc universally induces DNA damage in all six organs examined. Either inhibiting O-GlcNAcylation or supplementing dNTP pool diminishes the induced DNA damage in these organs, indicating that the mechanism of action is similar to that of high glucose treatment in pancreatic cells. Taken together, these results suggest the potential hazards of high sugar drinks and high glucosamine intake to genomic instability and possibly cancer initiation.

Homophilic ATP1A1 binding induces activin A secretion to promote EMT of tumor cells and myofibroblast activation.

Tumor cells with diverse phenotypes and biological behaviors are influenced by stromal cells through secretory factors or direct cell-cell contact. Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia with fibroblasts as the major cell type. In the present study, we observe enrichment of myofibroblasts in a juxta-tumoral position with tumor cells undergoing epithelial-mesenchymal transition (EMT) that facilitates invasion and correlates with a worse clinical prognosis in PDAC patients. Direct cell-cell contacts forming heterocellular aggregates between fibroblasts and tumor cells are detected in primary pancreatic tumors and circulating tumor microemboli (CTM). Mechanistically, ATP1A1 overexpressed in tumor cells binds to and reorganizes ATP1A1 of fibroblasts that induces calcium oscillations, NF-κB activation, and activin A secretion. Silencing ATP1A1 expression or neutralizing activin A secretion suppress tumor invasion and colonization. Taken together, these results elucidate the direct interplay between tumor cells and bound fibroblasts in PDAC progression, thereby providing potential therapeutic opportunities for inhibiting metastasis by interfering with these cell-cell interactions.

Angiotensin-converting enzyme inhibitors and risk of age-related macular degeneration in individuals with hypertension.

AIMS: Several observational studies have examined the potential protective effect of angiotensin-converting enzyme inhibitor (ACE-I) use on the risk of age-related macular degeneration (AMD) and have reported contradictory results owing to confounding and time-related biases. We aimed to assess the risk of AMD in a base cohort of patients aged 40 years and above with hypertension among new users of ACE-I compared to an active comparator cohort of new users of calcium channel blockers (CCB) using data obtained from IQVIA Medical Research Data, a primary care database in the UK. METHODS: In this study, 53 832 and 43 106 new users of ACE-I and CCB were included between 1995 and 2019, respectively. In an on-treatment analysis, patients were followed up from the time of index drug initiation to the date of AMD diagnosis, loss to follow-up, discontinuation or switch to the comparator drug. A comprehensive range of covariates were used to estimate propensity scores to weight and match new users of ACE-I and CCB. Standardized mortality ratio weighted Cox proportional hazards model was used to estimate hazard ratios of developing AMD. RESULTS: During a median follow-up of 2 years (interquartile range 1-5 years), the incidence rate of AMD was 2.4 (95% confidence interval 2.2-2.6) and 2.2 (2.0-2.4) per 1000 person-years among the weighted new users of ACE-I and CCB, respectively. There was no association of ACE-I use on the risk of AMD compared to CCB use in either the propensity score weighted or matched, on-treatment analysis (adjusted hazard ratio: 1.07 [95% confidence interval 0.90-1.27] and 0.87 [0.71-1.07], respectively). CONCLUSION: We found no evidence that the use of ACE-I is associated with risk of AMD in patients with hypertension.

Emerging therapies and their delivery for treating age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness in the Western world and is characterised in its latter stages by retinal cell death and neovascularisation and earlier stages with the loss of parainflammatory homeostasis. Patients with neovascular AMD (nAMD) are treated with frequent intraocular injections of anti-vascular endothelial growth factor (VEGF) therapies, which are not only unpopular with patients but carry risks of sight-threatening complications. A minority of patients are unresponsive with no alternative treatment available, and some patients who respond initially eventually develop a tolerance to treatment. New therapeutics with improved delivery methods and sustainability of clinical effects are required, in particular for non-neovascular AMD (90% of cases and no current approved treatments). There are age-related and disease-related changes that occur which can affect ocular drug delivery. Here, we review the latest emerging therapies for AMD, their delivery routes and implications for translating to clinical practice. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Characterization of initial key steps of IL-17 receptor B oncogenic signaling for targeted therapy of pancreatic cancer.

The members of the interleukin-17 (IL-17) cytokine family and their receptors were identified decades ago. Unlike IL-17 receptor A (IL-17RA), which heterodimerizes with IL-17RB, IL-17RC, and IL-17RD and mediates proinflammatory gene expression, IL-17RB plays a distinct role in promoting tumor growth and metastasis upon stimulation with IL-17B. However, the molecular basis by which IL-17RB promotes oncogenesis is unknown. Here, we report that IL-17RB forms a homodimer and recruits mixed-lineage kinase 4 (MLK4), a dual kinase, to phosphorylate it at tyrosine-447 upon treatment with IL-17B in vitro. Higher amounts of phosphorylated IL-17RB in tumor specimens obtained from patients with pancreatic cancer correlated with worse prognosis. Phosphorylated IL-17RB recruits the ubiquitin ligase tripartite motif containing 56 to add lysine-63-linked ubiquitin chains to lysine-470 of IL-17RB, which further assembles NF-κB activator 1 (ACT1) and other factors to propagate downstream oncogenic signaling. Consequentially, IL-17RB mutants with substitution at either tyrosine-447 or lysine-470 lose their oncogenic activity. Treatment with a peptide consisting of amino acids 403 to 416 of IL-17RB blocks MLK4 binding, tyrosine-477 phosphorylation, and lysine-470 ubiquitination in vivo, thereby inhibiting tumorigenesis and metastasis and prolonging the life span of mice bearing pancreatic tumors. These results establish a clear pathway of how proximal signaling of IL-17RB occurs and provides insight into how this pathway provides a therapeutic target for pancreatic cancer.

Redox sensor NPGPx restrains ZAP70 activity and modulates T cell homeostasis.

Emerging evidences implicate the contribution of ROS to T cell activation and signaling. The tyrosine kinase, ζ-chain-associated protein of 70 kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we show that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, results in T cell hyperproliferation and elevated cytokine productions. T cell-specific NPGPx-knockout mice reveal enhanced T-dependent humoral responses and are susceptible to experimental autoimmune encephalomyelitis (EAE). Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. NPGPx is activated by ROS generated from TCR stimulation, and modulates ZAP70 activity through redox switching to reduce ZAP70 recruitment to TCR/CD3 complex in membrane lipid raft, therefore subduing TCR responses. These results reveal a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.

Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis.

Human caspase-4 and its mouse homolog caspase-11 are receptors for cytoplasmic lipopolysaccharide. Activation of the caspase-4/11-dependent NLRP3 inflammasome is required for innate defense and endotoxic shock, but how caspase-4/11 is modulated remains unclear. Here, we show that mice lacking the oxidative stress sensor glutathione peroxidase 8 (GPx8) are more susceptible to colitis and endotoxic shock, and exhibit reduced richness and diversity of the gut microbiome. C57BL/6 mice that underwent adoptive cell transfer of GPx8-deficient macrophages displayed a similar phenotype of enhanced colitis, indicating a critical role of GPx8 in macrophages. GPx8 binds covalently to caspase-4/11 via disulfide bonding between cysteine 79 of GPx8 and cysteine 118 of caspase-4 and thus restrains caspase-4/11 activation, while GPx8 deficiency leads to caspase-4/11-induced inflammation during colitis and septic shock. Inhibition of caspase-4/11 activation with small molecules reduces the severity of colitis in GPx8-deficient mice. Notably, colonic tissues from patients with ulcerative colitis display low levels of Gpx8 and high caspase-4 expression. In conclusion, these results suggest that GPx8 protects against colitis by negatively regulating caspase-4/11 activity.

An open toolkit for tracking open science partnership implementation and impact.

Serious concerns about the way research is organized collectively are increasingly being raised. They include the escalating costs of research and lower research productivity, low public trust in researchers to report the truth, lack of diversity, poor community engagement, ethical concerns over research practices, and irreproducibility. Open science (OS) collaborations comprise of a set of practices including open access publication, open data sharing and the absence of restrictive intellectual property rights with which institutions, firms, governments and communities are experimenting in order to overcome these concerns. We gathered two groups of international representatives from a large variety of stakeholders to construct a toolkit to guide and facilitate data collection about OS and non-OS collaborations. Ultimately, the toolkit will be used to assess and study the impact of OS collaborations on research and innovation. The toolkit contains the following four elements: 1) an annual report form of quantitative data to be completed by OS partnership administrators; 2) a series of semi-structured interview guides of stakeholders; 3) a survey form of participants in OS collaborations; and 4) a set of other quantitative measures best collected by other organizations, such as research foundations and governmental or intergovernmental agencies. We opened our toolkit to community comment and input. We present the resulting toolkit for use by government and philanthropic grantors, institutions, researchers and community organizations with the aim of measuring the implementation and impact of OS partnership across these organizations. We invite these and other stakeholders to not only measure, but to share the resulting data so that social scientists and policy makers can analyse the data across projects.

NPGPx-Mediated Adaptation to Oxidative Stress Protects Motor Neurons from Degeneration in Aging by Directly Modulating O-GlcNAcase.

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, usually occurs in middle-aged people. However, the molecular basis of age-related cumulative stress in ALS pathogenesis remains elusive. Here, we found that mice deficient in NPGPx (GPx7), an oxidative stress sensor, develop ALS-like phenotypes, including paralysis, muscle denervation, and motor neurons loss. Unlike normal spinal motor neurons that exhibit elevated O-GlcNAcylation against age-dependent oxidative stress, NPGPx-deficient spinal motor neurons fail to boost O-GlcNAcylation and exacerbate ROS accumulation, leading to cell death. Mechanistically, stress-activated NPGPx inhibits O-GlcNAcase (OGA) through disulfide bonding to fine-tune global O-GlcNAcylation. Pharmacological inhibition of OGA rescues spinal motor neuron loss in aged NPGPx-deficient mice. Furthermore, expression of NPGPx in ALS patients is significantly lower than in unaffected adults. These results suggest that NPGPx modulates O-GlcNAcylation by inhibiting OGA to cope with age-dependent oxidative stress and protect motor neurons from degeneration, providing a potential therapeutic axis for ALS.

Role of protein structure in variant annotation: structural insight of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency.

Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH4) cofactor biosynthesis, and its deficiency represents the most common form of BH4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms. We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools.

High Glucose Triggers Nucleotide Imbalance through O-GlcNAcylation of Key Enzymes and Induces KRAS Mutation in Pancreatic Cells.

KRAS mutations are the earliest events found in approximately 90% of pancreatic ductal adenocarcinomas (PDACs). However, little is known as to why KRAS mutations preferentially occur in PDACs and what processes/factors generate these mutations. While abnormal carbohydrate metabolism is associated with a high risk of pancreatic cancer, it remains elusive whether a direct relationship between KRAS mutations and sugar metabolism exists. Here, we show that under high-glucose conditions, cellular O-GlcNAcylation is significantly elevated in pancreatic cells that exhibit lower phosphofructokinase (PFK) activity than other cell types. This post-translational modification specifically compromises the ribonucleotide reductase (RNR) activity, leading to deficiency in dNTP pools, genomic DNA alterations with KRAS mutations, and cellular transformation. These results establish a mechanistic link between a perturbed sugar metabolism and genomic instability that induces de novo oncogenic KRAS mutations preferentially in pancreatic cells.