Short Caco-2 model for evaluation of drug permeability: A sodium valerate-assisted approach.

The human colorectal adenocarcinoma cell line Caco-2, widely used for studying intestinal drug permeability, is typically grown on permeable filter supports and matures in 21 days with frequent media changes. The process is labor-intensive, prone to contamination, and has low throughput, contributing to the overall high utilization cost. Efforts to establish a low-cost, high-throughput, and short-duration model have encountered obstacles, such as weaker tight junctions causing monolayer leaks, incomplete differentiation resulting in low transporter expression, intricate and challenging protocols, and cytotoxicity, limiting the usability. Hence, this study aimed to develop a low-cost, efficient, and short-duration model by addressing the aforementioned concerns by customizing the media and finding a safe differentiation inducer. We generated a new rapid model using sodium valerate, which demonstrated sufficient transporter activity, improved monolayer integrity, and higher levels of differentiation markers than the 21-day model. Furthermore, this model exhibited consistent and reliable results when used to evaluate drug permeability over multiple days of repeated use. This study demonstrates the potential of a sodium valerate-assisted abbreviated model for drug permeability assessment with economic and practical advantages.

Emerging Immune Checkpoint Molecules on Cancer Cells: CD24 and CD200.

Cancer immunotherapy strategies are based on the utilization of immune checkpoint inhibitors to instigate an antitumor immune response. The efficacy of immune checkpoint blockade, directed at adaptive immune checkpoints, has been demonstrated in select cancer types. However, only a limited subset of patients has exhibited definitive outcomes characterized by a sustained response after discontinuation of therapy. Recent investigations have highlighted the significance of immune checkpoint molecules that are overexpressed in cancer cells and inhibit myeloid lineage immune cells within a tumor microenvironment. These checkpoints are identified as potential targets for anticancer immune responses. Notably, the immune checkpoint molecules CD24 and CD200 have garnered attention owing to their involvement in tumor immune evasion. CD24 and CD200 are overexpressed across diverse cancer types and serve as signaling checkpoints by engaging their respective receptors, Siglec-10 and CD200 receptor, which are expressed on tumor-associated myeloid cells. In this review, we summarized and discussed the latest advancements and insights into CD24 and CD200 as emergent immune checkpoint moieties, further delving into their therapeutic potentials for cancer treatment.

Structural basis for the substrate specificity of an S-formylglutathione hydrolase derived from Variovorax sp. PAMC 28711.

S-Formylglutathione hydrolase was originally known to catalyze the hydrolysis of S-formylglutathione to formate and glutathione. However, this enzyme has a broader esterase activity toward substrates containing thioester and ester bonds. In a previous study, we identified a new S-formylglutathione hydrolase (VaSFGH) gene in the Antarctic bacterium Variovorax sp. PAMC 28711, and recombinant VaSFGH protein was purified and characterized. Previous enzyme activity assays showed that VaSFGH has high activity, especially toward short-chain p-nitrophenyl esters (C2-C4). In this study, we determined the crystal structure of substrate-free VaSFGH at a resolution of 2.38 Å. In addition, p-nitrophenyl ester-bound VaSFGH structure models were generated by molecular docking simulations to obtain structural evidence of its substrate specificity. Comparative structural analysis of the apo-form and p-nitrophenyl ester-bound VaSFGH model structures revealed that large substrates could not bind inside the hydrophobic substrate-binding pocket because of the intrinsically static and relatively small substrate-binding pocket size of VaSFGH. This study provides useful information for further protein engineering of SFGHs for industrial use.

BAI1 nuclear expression reflects the survival of nonsmoking non-small cell lung cancer patients.

BACKGROUND: Smoking- and nonsmoking-associated lung cancers have different mechanisms of carcinogenesis. We divided non-small cell lung cancer (NSCLC) patients into nonsmoking and smoking groups with the aim of trying to understand the utility of brain-specific angiogenesis inhibitor 1 (BAI1) expression in the separate groups. METHODS: Clinicopathological data were obtained from 148 patients who had undergone surgery for NSCLC of the lung. Tissue microarray blocks were made of samples from NSCLC patients. Two pathologists graded the intensity of BAI1 expression as high or low expression in the cancer cells of patients in the smoking and nonsmoking groups. RESULTS: NSCLC nonsmokers with higher BAI1 nuclear expression had poor disease-specific survival (DSS) (hazard ratio: 2.679; 95% confidence interval [CI]:1.022-7.022, p = 0.045). The Kaplan-Meier survival curve confirmed that higher BAI1 expression was significantly associated with poor DSS (p = 0.034) in the nonsmoking group. CONCLUSIONS: We divided NSCLC patients into nonsmoking and smoking groups and found that nuclear BAI1 expression was related to patient survival in nonsmoking NSCLC patients. We suggest BAI1 expression as a predictive marker of nonsmoking-associated NSCLC and recommend that it be evaluated as an AJCC staging criterion in the future.

Enzymatic reaction mechanism of cis-aconitate decarboxylase based on the crystal structure of IRG1 from Bacillus subtilis.

Itaconate, which is formed by decarboxylation of cis-aconitate-an intermediate metabolite in the tricarboxylic acid cycle-has been used as a building block in polymer synthesis and is an important chemical in several biomedical and industrial applications. Itaconate is an immunometabolite with antibacterial, antiviral, immunoregulatory, and tumor-promoting activities. Recent focus has been on the role of itaconate in the field of immunology, with immune-responsive gene 1 (IRG1) being identified as the cis-aconitate decarboxylase responsible for itaconate production. We solved the structure of IRG1 from Bacillus subtilis (bsIRG1) and showed that IRG1 adopts either a closed or an open conformation; bsIRG1 was in the open form. A1 and A2 loops around the active site are flexible and can control the formation of the open and closed forms of IRG1. An in silico docking simulation showed that only the open form of IRG1 can accommodate the substrate. The most energetically favorable position of cis-aconitate in the active site of bsIRG1 involved the localization of C2 and C5 of cis-aconitate into the H102 region and H151 region of bsIRG1, respectively. Based on the structural study of bsIRG1, compared with IDS epimerase, and in silico docking simulation, we proposed two tentative enzymatic reaction mechanisms of IRG1, a two-base model and a one-base model.

A Family Affected by Novel C213W Mutation in PRPH2: Long-Term Follow-Up of CNV Secondary to Pattern Dystrophy.

The authors describe a family of three related individuals with a previously unreported mutation in the PRPH2 gene (C213W), which is associated with pattern dystrophy simulating fundus flavimaculatus. Four eyes later developed exudative maculopathy with choroidal neovascularization, which required injections of intravitreal anti-vascular endothelial growth factor (VEGF). This study reports the effectiveness of anti-VEGF therapy and long-term follow-up data in a family with a C213W mutation in the PRPH2 gene. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:354-362.].

Phosphatidylserine on viable sperm and phagocytic machinery in oocytes regulate mammalian fertilization.

Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm:egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAI1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAI1/BAI3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAI1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm:egg fusion.

Apoptotic cell clearance in the tumor microenvironment: a potential cancer therapeutic target.

Millions of cells in the human body undergo apoptosis not only under normal physiological conditions but also under pathological conditions such as infection or other diseases related to acute tissue injury. Swift apoptotic cell clearance is essential for tissue homeostasis. Defective clearance of dead cells is linked to pathogenesis of diseases such as inflammatory diseases, atherosclerosis, neurological disease, and cancer. Significance of apoptotic cell clearance has been emerging as an interesting field for disease treatment. Efficient apoptotic cell clearance plays an important role in reducing inflammation through the suppression of inappropriate inflammatory responses under healthy and diseased conditions. However, apoptotic cell clearance related to cancer pathogenesis is more complex in tumor microenvironments. Chronic inflammation resulting from the failure of apoptotic cell clearance can contribute to tumor progression. Conversely, tumor cells can exploit the anti-inflammatory effect of apoptotic cell clearance to generate an immunosuppressive tumor microenvironment. In this review, focus is on the current understanding of apoptotic cell clearance in the tumor microenvironment. Furthermore, we discuss how signaling molecules (PtdSer and PtdSer recognition receptor) mediating apoptotic cell clearance are aberrantly expressed in the tumor microenvironment and their current development state as potential therapeutic targets for clinical cancer therapy.

Predicting visual function after an ocular bee sting.

PURPOSE: To report a case of toxic optic neuropathy caused by an ocular bee sting. METHODS: Case report and literature review. RESULTS: A 44-year-old female presented with no light perception vision 2 days after a corneal bee sting in her right eye. She was found to have diffuse cornea edema with overlying epithelial defect and a pinpoint penetrating laceration at 6 o'clock. There was an intense green color to the cornea. The pupil was fixed and dilated with an afferent pupillary defect. A small hyphema was seen, and a dense white cataract had formed. A diagnosis of toxic endophthalmitis with associated toxic optic neuropathy was made. The patient underwent pars plana vitrectomy and lensectomy with anterior chamber washout. She was also placed on systemic broad-spectrum antibiotics. She had noted clinical improvement over the course of her hospitalization and was discharged with light perception vision. A corneal opacity precluded viewing of the fundus. We utilized ganzfeld electroretinography and flash visual evoked potentials (2 and 10 Hz) to assess the visual function. Both tests were normal and predicted improvement following restorative surgery. She underwent a secondary lens implantation with penetrating keratoplasty 7 months later. This was followed by an epiretinal membrane peel 1 year after the bee sting. Her best corrected visual acuity improved to 20/80. CONCLUSION: Toxic endophthalmitis and toxic optic neuropathy can be complications of ocular bee sting. We discuss the management of this rare occurrence and the role of electroretinographic testing and visual evoked potentials in predicting visual outcome.

Understanding the Role of the BAI Subfamily of Adhesion G Protein-Coupled Receptors (GPCRs) in Pathological and Physiological Conditions.

Brain-specific angiogenesis inhibitors (BAIs) 1, 2, and 3 are members of the adhesion G protein-coupled receptors, subfamily B, which share a conserved seven-transmembrane structure and an N-terminal extracellular domain. In cell- and animal-based studies, these receptors have been shown to play diverse roles under physiological and pathological conditions. BAI1 is an engulfment receptor and performs major functions in apoptotic-cell clearance and interacts (as a pattern recognition receptor) with pathogen components. BAI1 and -3 also participate in myoblast fusion. Furthermore, BAI1⁻3 have been linked to tumor progression and neurological diseases. In this review, we summarize the current understanding of the functions of BAI1⁻3 in pathological and physiological conditions and discuss future directions in terms of the importance of BAIs as pharmacological targets in diseases.

Structure-Based Classification and Anti-Cancer Effects of Plant Metabolites.

A variety of malignant cancers affect the global human population. Although a wide variety of approaches to cancer treatment have been studied and used clinically (surgery, radiotherapy, chemotherapy, and immunotherapy), the toxic side effects of cancer therapies have a negative impact on patients and impede progress in conquering cancer. Plant metabolites are emerging as new leads for anti-cancer drug development. This review summarizes these plant metabolites with regard to their structures and the types of cancer against which they show activity, organized by the organ or tissues in which each cancer forms. This information will be helpful for understanding the current state of knowledge of the anti-cancer effects of various plant metabolites against major types of cancer for the further development of novel anti-cancer drugs.

Context-dependent compensation among phosphatidylserine-recognition receptors.

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk -/- Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk -/- mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk +/+ and Mertk -/- mice. Prior to the onset of photoreceptor degeneration, Mertk -/- mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

The Role of Kinase Modulators in Cellular Senescence for Use in Cancer Treatment.

Recently, more than 30 small molecules and eight monoclonal antibodies that modulate kinase signaling have been approved for the treatment of several pathological conditions, including cancer, idiopathic pulmonary fibrosis, and rheumatoid arthritis. Among them, kinase modulators have been a primary focus for use in cancer treatment. Cellular senescence is believed to protect cells from tumorigenesis by irreversibly halting cell cycle progression and avoiding the growth of damaged cells and tissues. Senescence can also contribute to tumor suppression and be utilized as a mechanism by anti-cancer agents. Although the role of kinase modulators in cancer treatment and their effects on senescence in tumor development have been extensively studied, the relationship between kinase modulators for cancer treatment and senescence has not been fully discussed. In this review, we discuss the pro- and anti-tumorigenesis functions of senescence and summarize the key roles of kinase modulators in the regulation of senescence against tumors.

Structural aspects of transglutaminase 2: functional, structural, and regulatory diversity.

Transglutaminase 2 (TG2) is a multi-functional protein that has both protein cross-linking and guanosine 5'-triphosphate (GTP) hydrolysis activities. The activities of this protein are controlled by many cellular factors, including calcium (Ca2+) and GTP, and have been implicated in several physiological activities, including apoptosis, angiogenesis, wound healing, cellular differentiation, neuronal regeneration, and bone development. TG2 is linked to many human diseases such as inflammatory disease, celiac disease, neurodegenerative disease, diabetes, tissue fibrosis, and various cancers and is one of the most dynamic enzymes in terms of its functions, structures, and regulatory mechanisms. The aim of this review was to summarize the functional, structural, and regulatory diversity of TG2, with a particular focus on the structure of TG2.

Adhesion GPCRs as Modulators of Immune Cell Function.

Immune cells express several adhesion G protein-coupled receptors (aGPCRs), including the ADGRE subfamily members EMR1 (F4/80, ADGRE1), EMR2 (ADGRE2), EMR3 (ADGRE3), EMR4 (FIRE, ADGRE4), and CD97 (ADGRE5), the ADGRB subfamily member BAI1 (ADGRB1), and the ADGRG subfamily members GPR56 (ADGRG1), GPR97 (Pb99, ADGRG3), and GPR114 (ADGRG5). Expression of these molecules in hematopoietic stem and progenitor cells, monocytes/macrophages (Mφs), dendritic cells, granulocytes, and lymphocytes depends on lineage diversification and maturation, making them suitable markers for individual leukocyte subsets (e.g., F4/80 on mouse Mφs). Recent studies revealed intriguing activities of aGPCRs in tolerance induction (EMR1), granulopoiesis (CD97), engulfment of apoptotic cells and bacteria (BAI1), hematopoietic stem cell formation (GPR56), and control of cytotoxicity (GPR56). Here, we review these findings and discuss their biological and translational implications.

Genetic requirement for Mycl and efficacy of RNA Pol I inhibition in mouse models of small cell lung cancer.

Small cell lung cancer (SCLC) is a devastating neuroendocrine carcinoma. MYCL (L-Myc) is frequently amplified in human SCLC, but its roles in SCLC progression are poorly understood. We isolated preneoplastic neuroendocrine cells from a mouse model of SCLC and found that ectopic expression of L-Myc, c-Myc, or N-Myc conferred tumor-forming capacity. We focused on L-Myc, which promoted pre-rRNA synthesis and transcriptional programs associated with ribosomal biogenesis. Deletion of Mycl in two genetically engineered models of SCLC resulted in strong suppression of SCLC. The high degree of suppression suggested that L-Myc may constitute a therapeutic target for a broad subset of SCLC. We then used an RNA polymerase I inhibitor to target rRNA synthesis in an autochthonous Rb/p53-deleted mouse SCLC model and found significant tumor inhibition. These data reveal that activation of RNA polymerase I by L-Myc and other MYC family proteins provides an axis of vulnerability for this recalcitrant cancer.

Boosting Apoptotic Cell Clearance by Colonic Epithelial Cells Attenuates Inflammation In Vivo.

Few apoptotic corpses are seen even in tissues with high cellular turnover, leading to the notion that the capacity for engulfment in vivo is vast. Whether corpse clearance can be enhanced in vivo for potential benefit is not known. In a colonic inflammation model, we noted that the expression of the phagocytic receptor Bai1 was progressively downmodulated. Consistent with this, BAI1-deficient mice had more pronounced colitis and lower survival, with many uncleared apoptotic corpses and inflammatory cytokines within the colonic epithelium. When we engineered and tested transgenic mice overexpressing BAI1, these had fewer apoptotic cells, reduced inflammation, and attenuated disease. Boosting BAI1-mediated uptake by intestinal epithelial cells (rather than myeloid cells) was important in attenuating inflammation. A signaling-deficient BAI1 transgene could not provide a similar benefit. Collectively, these complementary genetic approaches showed that cell clearance could be boosted in vivo, with potential to regulate tissue inflammation in specific contexts.

Loss of phospholipase D2 impairs VEGF-induced angiogenesis.

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and critical for normal embryonic development and repair of pathophysiological conditions in adults. Although phospholipase D (PLD) activity has been implicated in angiogenic processes, its role in VEGF signaling during angiogenesis in mammals is unclear. Here, we found that silencing of PLD2 by siRNA blocked VEGF-mediated signaling in immortalized human umbilical vein endothelial cells (iHUVECs). Also, VEGF-induced endothelial cell survival, proliferation, migration, and tube formation were inhibited by PLD2 silencing. Furthermore, while Pld2-knockout mice exhibited normal development, loss of PLD2 inhibited VEGF-mediated ex vivo angiogenesis. These findings suggest that PLD2 functions as a key mediator in the VEGF-mediated angiogenic functions of endothelial cells. [BMB Reports 2016; 49(3): 191-196].

Clinical and visual outcomes of patients with uveitis in the mid-Atlantic United States.

PURPOSE: To report the clinical outcomes of uveitis patients at the University of Virginia. METHODS: Retrospective, observational study of uveitis patients seen at the University of Virginia from 1984 to 2014. Parametric and nonparametric methods were used to analyze the change in best-corrected visual acuity (BCVA) in relation to demographics, diagnoses, management, and complications. RESULTS: The study included 644 eyes of 491 patients. Patients with mild visual loss (logMAR <0.4) at presentation were younger than those with severe visual loss (SVL, logMAR >1.0) (P=0.002). Females were more likely to have mild visual loss as compared to males (P=0.025). Median overall BCVA was logMAR 0.18 at initial and final presentation (P=1.00). Vision loss at diagnosis was a predictor for moderate visual loss (MVL, logMAR 0.4 to <1.0) to SVL at last follow-up (P<0.001). Eyes with ocular hypertension were positively associated with MVL and SVL as compared to normotensive eyes (1.89 times at baseline, 2.62 times at last follow-up). Median BCVA was 0.18 logMAR for the anterior uveitis (AU) and 0.48 logMAR for the non-AU patients (P<0.001). AU patients were less likely to have SVL than non-AU group (P<0.001). AU group received local corticosteroids more frequently and systemic corticosteroids less commonly than non-AU patients (P<0.001). AU patients with MVL to SVL were more likely to have ophthalmic surgery (cataract, glaucoma or pars plana vitrectomy [PPV]) than those without MVL or SVL (P<0.001). Non-AU patients with MVL to SVL were more likely to have PPV than those without MVL or SVL (P=0.001). CONCLUSION: Mean overall BCVA remained stable. Favorable visual results were associated with younger age, female gender, and AU. Poor visual prognosis was concomitant with SVL at presentation and ocular hypertension. Ocular surgery (cataract extraction and glaucoma filtration) was more frequently performed for AU patients with MVL to SVL than those AU patients who did not experience moderate to SVL. PPV was commonly performed for both AU and non-AU patients with MVL to SVL.

Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1.

Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane-initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell-initiated pathway functioned independently of the liver X receptor (LXR) sterol-sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo.