Retinal pigment epithelium-specific ablation of GPx4 in adult mice recapitulates key features of geographic atrophy in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the elderly population, particularly the late-stage of dry AMD known as geographic atrophy (GA), lacks effective treatment options. Genetic mouse models of AMD have revealed the significance of impaired lipid metabolism and anti-oxidative capacity in early/intermediate stage of AMD, but remains unclear in GA that severely damages visual function. Here, to investigate the potential relevance of peroxidized lipids in RPE for late-stage dry AMD, GPx4fl/fl mice underwent subretinal injections of RPE-specific AAV-Cre vector or control AAV vector. RPE-specific GPx4 deficiency led to rapid RPE degeneration resembling key features of late-stage dry AMD, including preceding loss of RPE cell polarity, accumulation of acrolein, malondialdehyde, and 4-hydroxynonenal, photoreceptor loss, lipofuscin-laden subretinal melanophage infiltration, and complement activation. Treatment with α-tocopherol and ferrostatin-1 mitigated RPE degeneration, and shrunk mitochondria were observed in GPx4 deficient mice, suggesting involvement of ferroptosis. Unexpectedly, necrostatin-1s, an inhibitor of necroptosis, also ameliorated RPE degeneration, and activation of RIP3 and MLKL along with inactivation of caspase-8 was observed, indicating crosstalk between ferroptosis and necroptosis pathways. Our findings shed light on the intricate mechanisms underlying RPE degeneration in AMD and highlight GPx4/lipid peroxidation as potential therapeutic targets. RPE-specific ablation of GPx4 in mice provides a valuable tool for further elucidating the interplay between lipid peroxidation, cell death pathways, and AMD pathogenesis, offering new insights for preclinical research and therapeutic development targeting GA.

Significance of resected stomach measurements in postoperative delayed gastric emptying following laparoscopic pylorus-preserving gastrectomy.

PURPOSE: We investigated the relationship between the resected stomach measurements, the incidence of delayed gastric emptying (DGE), and food residue 1 year after surgery in patients who underwent laparoscopic pylorus-preserving gastrectomy (PPG). MATERIALS AND METHODS: The DGE group included 10 patients fasting due to nausea, vomiting, abdominal distension, or remnant stomach distension on radiographs; the control group included 36 patients without these symptoms. We compared the size and length of lesser and greater curvatures of the resected stomach and endoscopic findings after 1 year. RESULTS: No significant differences were observed between groups in terms of sex, body mass index, gross type, histology, tumor progression, number of dissected lymph nodes, operating time, or blood loss. The DGE group was older, had a longer postoperative stay, and showed a smaller size and shorter greater curvature of the resected stomach than the control group (p < 0.01 for all). No difference was observed in the length of the lesser curvature of the resected stomach. In addition, there were no disparities in residual food, degree and extent of gastritis, or bile reflux 1 year after gastrectomy. CONCLUSIONS: Measurements of the resected stomach suggest that preventing DGE may be achievable by removing a larger area of the greater curvature and/or stomach during laparoscopic PPG. This implies potential surgical strategy improvements for better outcomes. Further multicenter trials are needed to validate and refine techniques.

The NRF2 inducer CDDO-2P-Im provokes a reduction in amyloid ß levels in Alzheimer's disease model mice.

Alzheimer's disease (AD) is the most common etiology of dementia. The transcription factor NF-E2-related factor 2 (NRF2) induces the expression of genes encoding phase II detoxification and antioxidant genes. NRF2 is regulated by Kelch-like ECH-associated protein 1 (KEAP1), and the KEAP1-NRF2 system is the key regulatory system involved in cytoprotection. To examine whether pharmacological induction of NRF2 expression alleviates AD phenotypes in vivo, we employed two AD mouse models, i.e., App  NL-G-F/NL-G-F (AppNLGF) and APPV717I::TAUP301L (APP/TAU) mice. As the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11-dien-28-oyl)] (CDDO)-4(-pyridin-2-yl)-imidazole (CDDO-2P-Im) exhibits strong NRF2-inducing activity, we treated AD model mice with CDDO-2P-Im. We found that Aß42 levels were markedly greater in the brains of AppNLGF mice than in those of APP/TAU mice. CDDO-2P-Im treatment significantly decreased Aß42 levels, but not Aß40 levels, in APP/TAU mice. Consequently, CDDO-2P-Im also decreased the ratio of Aß42/Aß40, a vital marker of amyloid plaque formation. LC-MS/MS analyses revealed that CDDO-2P-Im was delivered to the brains of the APP/TAU mice. CDDO-2P-Im induced the expression of detoxification and antioxidant gene targets of NRF2 and elevated reduced glutathione (GSH) levels in the mouse brain. These results support the notion that CDDO-2P-Im ameliorates AD-related pathologic changes.

Specific cancer types and prognosis in patients with variations in the KEAP1-NRF2 system: A retrospective cohort study.

The KEAP1-NRF2 system induces the expression of antioxidant genes in response to various types of oxidative stress. Some cancer cells activate this system, which increases their malignancy through genetic mutations. We performed a retrospective cohort study using the C-CAT database, which contains the gene-panel sequence data from 60,056 cases of diagnosed solid tumors. We analyzed somatic mutations in NRF2 and KEAP1 genes and their associations with clinical outcomes. Variants in the NRF2 gene were clustered in exon 2, which encodes the DLG and ETGE motifs essential for KEAP1 interaction. The NRF2 variants were frequently observed in esophageal and lung squamous cell carcinoma with frequencies of 35.9% and 19.6%, respectively. Among these mutations, the NRF2 variants in the ETGE motif were indicators of a worse prognosis. KEAP1 variants were found in 2.5% of all cases. The variants were frequent in lung cancer and showed a worse prognosis in lung and other types of adenocarcinomas. We then conducted gene expression analysis using TCGA data. While cancers with DLG and ETGE variants were similar in terms of gene expression profiles, there were significant differences between cancers with KEAP1 and NRF2 variants. Our results indicate that genetic alteration of the KEAP1-NRF2 pathway is a major factor in patient prognosis for each cancer type and its genetic variant. Variants in NRF2 and KEAP1 genes can characterize the biological basis of each cancer type and are involved in carcinogenesis, resistance to therapy, and other biological differences.

Sensor systems of KEAP1 uniquely detecting oxidative and electrophilic stresses separately In vivo.

In the KEAP1-NRF2 stress response system, KEAP1 acts as a sensor for oxidative and electrophilic stresses through formation of S-S bond and C-S bond, respectively. Of the many questions left related to the sensor activity, following three appear important; whether these KEAP1 sensor systems are operating in vivo, whether oxidative and electrophilic stresses are sensed by the similar or distinct systems, and how KEAP1 equips highly sensitive mechanisms detecting oxidative and electrophilic stresses in vivo. To address these questions, we conducted a series of analyses utilizing KEAP1-cysteine substitution mutant mice, conditional selenocysteine-tRNA (Trsp) knockout mice, and human cohort whole genome sequence (WGS) data. Firstly, the Trsp-knockout provokes severe deficiency of selenoproteins and compensatory activation of NRF2. However, mice lacking homozygously a pair of critical oxidative stress sensor cysteine residues of KEAP1 fail to activate NRF2 in the Trsp-knockout livers. Secondly, this study provides evidence for the differential utilization of KEAP1 sensors for oxidative and electrophilic stresses in vivo. Thirdly, theoretical calculations show that the KEAP1 dimer equips quite sensitive sensor machinery in which modification of a single molecule of KEAP1 within the dimer is sufficient to affect the activity. WGS examinations of rare variants identified seven non-synonymous variants in the oxidative stress sensors in human KEAP1, while no variant was found in electrophilic sensor cysteine residues, supporting the fail-safe nature of the KEAP1 oxidative stress sensor activity. These results provide valuable information for our understanding how mammals respond to oxidative and electrophilic stresses efficiently.

Visual prognostic factors following surgery for stage 3 idiopathic epiretinal membrane by Govetto classification.

PURPOSE: To investigate the visual prognosis of metamorphopsia in patients undergoing surgery for stage 3 idiopathic epiretinal membrane (ERM) by Govetto classification using preoperative optical coherence tomography (OCT) parameters. STUDY DESIGN: Retrospective clinical study. METHOD: This study included 45 eyes of 45 patients with a minimum follow-up period of 3 months. The best-corrected visual acuity (BCVA) and metamorphopsia score using the M-CHARTS were recorded. Central foveal thickness (CFT), inner nuclear layer thickness (INL), ectopic inner retinal layer thickness (EIFL), outer retinal layer thickness, disruption of the ellipsoid zone, cotton ball sign, and intraretinal cystoid changes were measured based on spectral domain OCT. Preoperative and postoperative values and conditions were compared, and correlations between the preoperative values or conditions and postoperative metamorphopsia scores or BCVA were analyzed. RESULTS: After surgery, the horizontal, vertical, and mean metamorphopsia scores, as well as BCVA, CFT, INL, and EIFL significantly improved (p < 0.001). Using multivariate analysis, only preoperative CFT was a significant explanatory parameter for both the postoperative horizontal metamorphopsia scores and mean values of the postoperative horizontal and vertical metamorphopsia scores (p = 0.019 and p = 0.011, respectively). Age (p = 0.011) and preoperative CFT (p = 0.026) were significant explanatory parameters of postoperative BCVA. CONCLUSION: Preoperative CFT significantly correlated with postoperative metamorphopsia in patients undergoing surgery for stage 3 idiopathic ERM. This finding might help surgeons predict postoperative visual outcomes and make timely surgical decisions.

Multiple metastases of human epidermal growth factor receptor 2­positive, hormone receptor­positive, pT1a pN0 breast cancer within 1 year after surgery: A case report.

Adjuvant chemotherapy is usually not considered for pT1a pN0 human epidermal growth factor receptor 2 (HER2)-positive breast cancer due to its low recurrence rate. The present report describes a case of pT1a hormone receptor-positive HER2-positive breast cancer with multiple recurrences in the axillary lymph nodes and liver within 1 year after radical surgery. A 58-year-old woman underwent left total mastectomy and sentinel lymph node biopsy for left breast cancer with pathological stage IA (pT1a pN0). The subtype corresponded to luminal B-like breast cancer with a nuclear grade of 3 and a Ki-67 labeling index of 37%. An aromatase inhibitor (letrozole) was planned to be administered for 5 years after surgery, but the patient was diagnosed with multiple liver and axillary lymph node metastases 11 months after surgery. After 1 year of chemotherapy (paclitaxel) in combination with anti-HER2 therapy (pertuzumab and trastuzumab), liver metastases resolved. A complete response of the liver lesion has been maintained 4 years after the anti-HER2 therapy initiation. The present case exhibited two poor prognostic factors: High Ki-67 labeling index and nuclear grade 3. Based on the 'Predict' tool, the present case would be expected to have a cancer-related mortality rate of 6% 10 years after surgery with adjuvant endocrine therapy. Although this value may be controversial for postoperative anti-HER2 therapy, the present case should not be considered to be a low-risk case. When the identification of high-risk pT1a pN0 HER2-positive breast cancer is possible, postoperative anti-HER2 therapy plus chemotherapy would be effective in decreasing the rate of recurrence.

Differential squamous cell fates elicited by NRF2 gain of function versus KEAP1 loss of function.

Clinical evidence has revealed that high-level activation of NRF2 caused by somatic mutations in NRF2 (NFE2L2) is frequently detected in esophageal squamous cell carcinoma (ESCC), whereas that caused by somatic mutations in KEAP1, a negative regulator of NRF2, is not. Here, we aspire to generate a mouse model of NRF2-activated ESCC using the cancer-derived NRF2L30F mutation and cancer driver mutant TRP53R172H. Concomitant expression of NRF2L30F and TRP53R172H results in formation of NRF2-activated ESCC-like lesions. In contrast, while squamous-cell-specific deletion of KEAP1 induces similar NRF2 hyperactivation, the loss of KEAP1 combined with expression of TRP53R172H does not elicit the formation of ESCC-like lesions. Instead, KEAP1-deleted cells disappear from the esophageal epithelium over time. These findings demonstrate that, while cellular NRF2 levels are similarly induced, NRF2 gain of function and KEAP1 loss of function elicits distinct fates of squamous cells. The NRF2L30F mutant mouse model developed here will be instrumental in elucidating the mechanistic basis leading to NRF2-activated ESCC.

Whole blood transcriptome analysis for age- and gender-specific gene expression profiling in Japanese individuals.

Whole blood transcriptome analysis is a valuable approachin medical research, primarily due to the ease of sample collection and the richness of the information obtained. Since the expression profile of individual genes in the analysis is influenced by medical traits and demographic attributes such as age and gender, there has been a growing demand for a comprehensive database for blood transcriptome analysis. Here, we performed whole blood RNA sequencing (RNA-seq) analysis on 576 participants stratified by age (20-30s and 60-70s) and gender from cohorts of the Tohoku Medical Megabank (TMM). A part of female segment included pregnant women. We did not exclude the globin gene family in our RNA-seq study, which enabled us to identify instances of hereditary persistence of fetal hemoglobin based on the HBG1 and HBG2 expression information. Comparing stratified populations allowed us to identify groups of genes associated with age-related changes and gender differences. We also found that the immune response status, particularly measured by neutrophil-to-lymphocyte ratio (NLR), strongly influences the diversity of individual gene expression profiles in whole blood transcriptome analysis. This stratification has resulted in a data set that will be highly beneficial for future whole blood transcriptome analysis in the Japanese population.

Cross-Limb Vascular Shunting for Traumatic Popliteal Artery Injury.

BACKGROUND: Popliteal artery injury (PAI) is a challenging trauma that requires prompt and accurate treatment since the probability of lower-limb amputation increases with the ischemic time. Intravascular shunting and cross-limb vascular shunting (CLS) are used as temporary vascular shunting (TVS) methods to shorten the ischemic time for limb vascular injury. CLS involves sending blood from an artery in a healthy body part to a peripheral vessel in an injured part to immediately resume blood flow to the injured limb. For closed injuries including PAI, CLS may be performed without exploring and identifying the arterial stumps and it enables early reperfusion to the ischemic limb. We report the case series of traumatic PAI treated using CLS and verify the usefulness of CLS. METHODS: All patients with traumatic PAI treated with CLS at our institution between August 2013 and December 2021 were included. Demographic and clinical patient characteristics were extracted from the medical records. Comorbid injuries, severity of acute limb ischemia based on the Rutherford grading scale, time from injury to reperfusion by CLS, time from injury to completion of artery, and the use of fasciotomy were investigated. As outcomes, we investigated the presence or absence of lower extremity amputation during the course of treatment. RESULTS: We used CLS as treatment for 5 cases with traumatic PAI. Based on the Rutherford grading scale for acute limb ischemia, there were one limb with grade 2B and 4 with grade 3. Amputation of the lower extremities was avoided except for 1 extremity in which arterial reconstruction was not achieved due to unexplained cardiac arrest during surgery. CONCLUSIONS: CLS enables early reperfusion of the injured limb and is effective as a TVS method for traumatic PAI with severe ischemia or soft tissue damage.

Effects of NRF2 polymorphisms on safety and efficacy of bardoxolone methyl: subanalysis of TSUBAKI study.

BACKGROUND: In the TSUBAKI study, bardoxolone methyl significantly increased measured and estimated glomerular filtration rates (GFR) in patients with multiple forms of chronic kidney disease (CKD), including Japanese patients with type 2 diabetes and stage 3-4 CKD. Since bardoxolone methyl targets the nuclear factor erythroid 2-related factor 2 pathway, this exploratory analysis of the TSUBAKI study investigated the impact of the regulatory single nucleotide polymorphism, rs6721961, on the effects of bardoxolone methyl. METHODS: Japanese patients aged 20-79 years with type 2 diabetes and stage 3-4 CKD were randomized to bardoxolone methyl 5-15 mg/day (titrated as tolerated) or placebo for 16 weeks. Genotype frequency, clinical characteristics, renal function, and adverse events were primarily assessed. RESULTS: Of 104 patients (bardoxolone methyl n = 55, placebo n = 49); 57% were genotype C/C, 32% C/A and 12% A/A. The frequency of the A/A genotype was higher among patients with diabetic kidney disease than in the general Japanese population (~ 5%). Measured and estimated GFRs increased from baseline in all genotypes receiving bardoxolone methyl. There were no significant differences between genotypes for safety parameters, including blood pressure, bodyweight, and levels of B-type natriuretic peptide, or in the type and frequency of adverse events, suggesting that the efficacy and safety of bardoxolone methyl are unaffected by the rs6721961 polymorphism-617 (C→A) genotype. CONCLUSIONS: Our approach of combining genome analysis with clinical trials for an investigational drug provides important and useful clues for exploring the efficacy and safety of the drug. TRIAL REGISTRATION: ClinicalTrials.gov; NCT02316821.

An easy-to-implement, non-invasive head restraint method for monkey fMRI.

Functional magnetic resonance imaging (fMRI) in behaving monkeys has a strong potential to bridge the gap between human neuroimaging and primate neurophysiology. In monkey fMRI, to restrain head movements, researchers usually surgically implant a plastic head-post on the skull. Although time-proven to be effective, this technique could create burdens for animals, including a risk of infection and discomfort. Furthermore, the presence of extraneous objects on the skull, such as bone screws and dental cement, adversely affects signals near the cortical surface. These side effects are undesirable in terms of both the practical aspect of efficient data collection and the spirit of "refinement" from the 3R's. Here, we demonstrate that a completely non-invasive fMRI scan in awake monkeys is possible by using a plastic head mask made to fit the skull of individual animals. In all of the three monkeys tested, longitudinal, quantitative assessment of head movements showed that the plastic mask has effectively suppressed head movements, and we were able to obtain reliable retinotopic BOLD signals in a standard retinotopic mapping task. The present, easy-to-make plastic mask has a strong potential to simplify fMRI experiments in awake monkeys, while giving data that is as good as or even better quality than that obtained with the conventional head-post method.

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation.

PURPOSE: To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. METHODS: EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. RESULTS: Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. CONCLUSION: SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

Preliminary Results of Branch Level, Brachial Plexus Peripheral Nerve Stimulation on a Non-Human Primate.

Restoring functional hand control is a priority for those suffering from neurological impairments. Functional electrical stimulation (FES) is commonly applied to assist with rehabilitation. However, FES applied intramuscularly typically results in complex surgeries requiring many implants. This paper presents the preliminary findings from a feasibility study focused on evaluating the potential to access the upper extremity peripheral nerves through a single surgical approach (axillary approach). A single Japanese macaque (macaca fuscata) monkey was used to validate the feasibility of this study. Four of the five peripheral nerves which control the upper extremity were exposed, and had multi-contact epineural cuffs implanted: median, radial, ulnar and musculocutaneous. The axillary nerve was not accessible for epineural cuff placement with the current surgical approach used in this study. Electrical stimuli were used to produce movement contraction patterns of muscles relevant to the innervated peripheral nerves. In addition, to assist in quantifying the outcome, evoked potentials were simultaneously recorded from five extrinsic forearm flexors during median nerve stimulation. This feasibility study demonstrated that the axillary approach enables electrode placement to four of the five peripheral nerves required for upper extremity control through a single skin incision.Clinical relevance- This study demonstrated that the electrode placement to most of the peripheral nerves that control the arm and hand can be done by a single surgical approach: axillary approach.

Optimal information loading into working memory explains dynamic coding in the prefrontal cortex.

Working memory involves the short-term maintenance of information and is critical in many tasks. The neural circuit dynamics underlying working memory remain poorly understood, with different aspects of prefrontal cortical (PFC) responses explained by different putative mechanisms. By mathematical analysis, numerical simulations, and using recordings from monkey PFC, we investigate a critical but hitherto ignored aspect of working memory dynamics: information loading. We find that, contrary to common assumptions, optimal loading of information into working memory involves inputs that are largely orthogonal, rather than similar, to the late delay activities observed during memory maintenance, naturally leading to the widely observed phenomenon of dynamic coding in PFC. Using a theoretically principled metric, we show that PFC exhibits the hallmarks of optimal information loading. We also find that optimal information loading emerges as a general dynamical strategy in task-optimized recurrent neural networks. Our theory unifies previous, seemingly conflicting theories of memory maintenance based on attractor or purely sequential dynamics and reveals a normative principle underlying dynamic coding.

Association of Subretinal Fluid Duration and Baseline Chorioretinal Structure With Optical Coherence Tomography in Central Serous Chorioretinopathy.

Purpose: This study investigated the association between subretinal fluid (SRF) duration and baseline chorioretinal structure in patients with naïve (first-episode and untreated) central serous chorioretinopathy (CSC). Methods: We divided 59 patients into non-prolonged (<3 months) and prolonged (>3 months) groups based on SRF fluid persistence from the initial visit. The follow-up period varied as the shorter duration was chosen between SRF disappearance time and 3 months from the initial visit. We measured the central retinal thickness (CRT), central choroidal thickness (CCT), SRF height (SRFH), and outer nuclear layer thickness (ONL) using spectral-domain optical coherence tomography (SD-OCT) at the initial visit and recorded SRF duration. We compared these parameters between the groups, conducted multivariate analysis for SRF duration of more than 3 months, and investigated the correlation among CCT and CRT, SRFH, or ONL, and among SRF duration and CRT, CCT, SRFH, or ONL. Results: CCT was significantly thicker in the prolonged than in the non-prolonged group at the initial visit (P = 0.044) and significantly correlated with CRT and SRFH (P = 0.007, r = 0.35 and P = 0.002, r = 0.39). SRF duration significantly correlated with CRT and SRFH (P = 0.009, r = 0.40 and P = 0.003, r = 0.41). The optimal model for SRF duration more than 3 months included age (P = 0.054) and CCT (P = 0.008). Conclusions: Thicker baseline CCT can lead to a longer SRF duration in naïve CSC. Translational Relevance: This association aids in early detection of cases at a higher risk of prolonged SRF.

Mapping information flow between the inferotemporal and prefrontal cortices via neural oscillations in memory retrieval and maintenance.

Interaction between the inferotemporal (ITC) and prefrontal (PFC) cortices is critical for retrieving information from memory and maintaining it in working memory. Neural oscillations provide a mechanism for communication between brain regions. However, it remains unknown how information flow via neural oscillations is functionally organized in these cortices during these processes. In this study, we apply Granger causality analysis to electrocorticographic signals from both cortices of monkeys performing visual association tasks to map information flow. Our results reveal regions within the ITC where information flow to and from the PFC increases via specific frequency oscillations to form clusters during memory retrieval and maintenance. Theta-band information flow in both directions increases in similar regions in both cortices, suggesting reciprocal information exchange in those regions. These findings suggest that specific subregions function as nodes in the memory information-processing network between the ITC and the PFC.