HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells.

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Novel ability of diflubenzuron as an inhibitor of mitochondrial function.

Compounds classified as benzoylphenylurea (BPU), such as diflubenzuron (DFB), are used as insecticides. Although BPU disrupts molting by inhibiting chitin biosynthesis and exhibits insecticidal activity, their exact mode of action remains unknown. Since epidermal cells proliferate and morphologically change from squamous to columnar cells during the early stages of insect molting, we speculate that a transition similar to that from epithelium to mesenchyme occurs and that BPU may inhibit this transition. Here, we addressed this possibility. We found that DFB decreases actin expression in insect cells (the tissue cultures of insect integument). Detailed analysis in Schneider S2 cells reveals that DFB inhibits the expression of actin isoforms (Act5C and Act42A) and the Drosophila ortholog of myocardin-related transcription factor (Mrtf), leading to cell growth suppression. Proteomics identified the Drosophila ortholog of prohibitin (Phb1D and Phb2E) as one of the DFB-binding proteins. DFB inhibits the interaction between Phb1D and Phb2E and induces mitochondrial dysfunction. The knock-down of Phb2E suppresses the expression of Act5C, Act42A, and Mrtf, leading to cell growth inhibition. Thus, the disruption of Phb function is a possible novel target of DFB.

Initial intravenous immunoglobulin therapy without aspirin for acute Kawasaki disease: a retrospective cohort study with a Bayesian inference.

OBJECTIVE: To clarify the necessity of acetylsalicylic acid (ASA) administration combined with intravenous immunoglobulin (IVIG) therapy in the treatment of acute Kawasaki disease. DESIGN: Retrospective cohort study. SETTING: Multicentre. PARTICIPANTS: This study included 735 patients with Kawasaki disease aged ≤10 years and hospitalised between 4 and 10 days of illness in eight Japanese hospitals from January 2016 to December 2020. EXPOSURES: High-dose (HD) ASA was administered with initial IVIG to 333 patients in 6 hospitals (HD group). ASA was not administered routinely to 402 patients in the other two hospitals, and low-dose ASA was only administered when patients developed coronary artery lesions or pericardial effusion (non-HD group). PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was the presence of coronary artery lesions, defined as a coronary artery diameter >+2.5 SD of body surface area within 1 month of onset. The secondary outcome was responsiveness to the initial IVIG therapy. Adjusted risk ratios for the outcomes were calculated using modified Poisson regression models. Bayesian analysis was conducted to estimate the posterior probability of the treatment effect of HD ASA under several prior distributions. RESULTS: The incidence of coronary artery lesions was not significantly higher in the HD group than in the non-HD group (12/333 (3.6%) vs 15/402 (4.0%)). The proportion of non-responders to initial IVIG was similar between the two groups (HD group: 78/333 (23%); non-HD group: 83/402 (22%)). In the Bayesian analysis, considering a difference of ≤2% to be of no clinical importance, there was only a 9.3% chance of reduced risk of coronary artery lesions in the HD group compared with the non-HD group even with a strongly enthusiastic prior for HD treatment. CONCLUSIONS: Compared with HD ASA treatment, treatment without ASA in the acute phase of Kawasaki disease was not associated with increased complications from Kawasaki disease.

High-risk factors for zonular complications during cataract surgery in eyes with pseudoexfoliation syndrome.

AIMS: To predict high-risk factors for zonular complications during cataract surgery due to pre-existing severe zonular dehiscence in eyes with pseudoexfoliation (PXF) syndrome. METHODS: 315 eyes of 315 consecutive patients with PXF scheduled for phacoemulsification surgery underwent preoperative examination of various ocular parameters using an anterior segment-optical coherence tomography and other devices. When zonular complications occurred during surgery due to zonular dehiscence, scleral fixation of the intraocular lens (IOL) or implantation of a capsular tension ring (CTR) was performed. High-risk factors for these intraoperative zonular complications were examined using classification-tree and logistic regression analyses. RESULTS: Of the 315 eyes, 31 (9.84%) underwent scleral IOL fixation or CTR implantation. High-risk factors identified by classification-tree analysis were a small pupillary diameter after mydriasis <6.30 mm, a shallow anterior chamber depth <2.074 mm and lens decentration >0.260 mm. Based on exact logistic regression analysis, the OR was 4.81-fold higher for eyes with poor mydriasis than for eyes without poor mydriasis (p=0.006, 95% CI 1.49 to 18.23), 23.99-fold higher for eyes with poor mydriasis and a shallow anterior chamber (p<0.001, 5.92 to 109.02) and 287.39-fold higher for eyes with poor mydriasis, a shallow chamber and great lens decentration (p<0.001, 50.46 to infinity). CONCLUSION: In eyes with PXF, high-risk factors for zonular complications during cataract surgery due to pre-existing severe zonular dehiscence were poor mydriasis, shallow anterior chamber and large lens decentration, suggesting the importance of evaluating these conditions preoperatively.

Isolation and propagation of bovine blood-derived macrophages using a mixed culture with bovine endothelial B46 cells.

Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.

Age-Related Changes in Total Corneal Astigmatism in Eyes With High Myopia.

PURPOSE: The purpose of this study was to compare age-related changes in corneal astigmatism in eyes with and without high myopia. METHODS: Eight-hundred eyes with high myopia (axial length ≥26.0 mm) and 800 eyes without high myopia (200 eyes each from patients in their 40s, 50s, 60s, and ≥70s) underwent videokeratographic examination. The amounts of vertical/horizontal (Rx) and oblique astigmatism (Ry) components, irregular astigmatism, and corneal shape were compared between eyes with and without high myopia and among age categories. RESULTS: In both groups, the mean Rx significantly changed to more positive with age (P < 0.001), whereas the Ry did not change significantly. The Rx was significantly more negative in the high myopia group than in the control group in all age categories (P ≤ 0.003), whereas the Ry did not differ significantly. The mean changes in the Rx and Ry during each 2 consecutive decades did not differ significantly between groups. The asymmetry and higher-order irregularity components increased with age (P ≤ 0.001) but did not differ significantly between groups, except for the higher-order irregularity in patients in their 60s (P = 0.018). In the averaged map, the corneal shape changed from with-the-rule to against-the-rule astigmatism with age in both groups, but the changes occurred later in the high myopia group. CONCLUSIONS: Age-related changes from with-the-rule to against-the-rule astigmatism occurred later in eyes with high myopia compared with eyes without high myopia in middle or older aged patients, but this change in each age decade was comparable between eyes with and without high myopia.

Incidence and causative agent distribution of viral-induced paediatric asthma exacerbations under strict infection control measures: a single-centre retrospective study in Japan.

BACKGROUND: The prevalence of respiratory viruses in children changed under strict infection control measures during the coronavirus disease 2019 (COVID-19) outbreak. In this study, we investigated the frequency of viral detection in the nasopharynx of paediatric patients with asthma exacerbations requiring hospitalization during the COVID-19 pandemic, as well as the distribution of causative viruses. METHODS: We included paediatric patients admitted for asthma exacerbations between November 2020 and December 2022 at a single centre in Kobe, Japan. Demographic, clinical, and laboratory data were collected from their medical records and using additional questionnaires. All patients enrolled in this study met the diagnostic criteria for asthma exacerbations outlined in the Japanese Pediatric Guideline for the Treatment and Management of Bronchial Asthma 2020. Statistical differences were calculated using univariate analyses (chi-square or Mann‒Whitney U test). RESULTS: We enrolled 203 children hospitalized for asthma attacks and collected nasopharyngeal samples from 189 patients. The median patient age was 3.0 years. Asthma severity was classified as mild (4.0%), moderate (82.3%), or severe (13.8%). The proportion of viral respiratory infections was 95.2% (180/189). The rate of patients with multiple viral infections was 20.6% (39/189). The most frequently detected pathogens were rhinovirus and enterovirus (RV/EV) at 69.3% (131/189), allowing for duplicate detection, followed by respiratory syncytial virus (RSV) at 28.6% (54/189). We also detected RV/EV almost every month compared to RSV and other viruses. In addition, RV/EV-positive patients were significantly older (p = 0.033), exhibited higher WBC counts (p < 0.001) and higher Eos counts (p < 0.001), had elevated total IgE levels (p < 0.001) and house dust mite-specific IgE levels (p = 0.019), had a shorter duration of hospitalization (p < 0.001), and had a shorter duration of oxygen therapy (p < 0.001). In patients positive for RV/EV, the use of ICSs significantly reduced the severity of the condition (p < 0.001). CONCLUSION: Even under strict infection control measures, respiratory viruses were detected in the nasopharynx of almost all paediatric patients who had asthma exacerbations requiring hospitalization.

Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

GH3-mediated auxin inactivation attenuates multiple stages of lateral root development.

Lateral root (LR) positioning and development rely on the dynamic interplay between auxin production, transport but also inactivation. Nonetheless, how the latter affects LR organogenesis remains largely uninvestigated. Here, we systematically analyze the impact of the major auxin inactivation pathway defined by GRETCHEN HAGEN3-type (GH3) auxin conjugating enzymes and DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) in all stages of LR development using reporters, genetics and inhibitors in Arabidopsis thaliana. Our data demonstrate that the gh3.1/2/3/4/5/6 hextuple (gh3hex) mutants display a higher LR density due to increased LR initiation and faster LR developmental progression, acting epistatically over dao1-1. Grafting and local inhibitor applications reveal that root and shoot GH3 activities control LR formation. The faster LR development in gh3hex is associated with GH3 expression domains in and around developing LRs. The increase in LR initiation is associated with accelerated auxin response oscillations coinciding with increases in apical meristem size and LR cap cell death rates. Our research reveals how GH3-mediated auxin inactivation attenuates LR development. Local GH3 expression in LR primordia attenuates development and emergence, whereas GH3 effects on pre-initiation stages are indirect, by modulating meristem activities that in turn coordinate root growth with LR spacing.

Morphology of the proximal tibiofibular joint and ligaments using three-dimensional computed tomography: A cadaveric study.

BACKGROUND: The aim of our study was to clarify the morphology of the proximal tibiofibular joint (PTFJ), insertion sites of the proximal tibiofibular ligaments (PTFLs), and related osseous landmarks on three-dimensional (3D) computed tomography (CT) images. METHODS: Cadaveric knees were evaluated by dissection and 3D CT imaging. The anterior PTFL (A-PTFL) and posterior PTFL (P-PTFL) were isolated, and their tibial and fibular insertion sites were identified. The morphology and location of insertion sites and their positional relationships with osseous structures were analyzed on 3D CT images. RESULTS: The A-PTFL comprised up to four bundles, and the P-PTFL comprised two bundles. The mean length of the A-PTFL and P-PTFL was 11.3 mm and 10.3 mm, respectively. On the tibial side of the PTFJ, bony prominences were present at the A-PTFL and P-PTFL insertion sites and were clearly identified as osseous landmarks in all knees. On the fibular side, the A-PTFL and P-PTFL insertion sites were at the edge of the triangular pyramid of the fibular head. The mean PTFJ area was 198.8 mm2, and the mean inclination angle between PTFJ and tibial plane was 38.4°. There was an inverse correlation between the PTFJ surface area and the inclination angle. CONCLUSION: The present study clearly identified PTFL insertion sites on the tibia and fibula and showed the relationships between these insertions and osseous landmarks. These data improve our understanding of the anatomy of PTFL insertions, which may assist surgeons in performing anatomical reconstruction.

Possible roles of bone morphogenetic protein 4 in regulating endometrial function in cows.

This study investigated the expression dynamics of bone morphogenetic protein 4 (BMP4) and its receptors (BMPR1A, BMPR1B, and BMPR2) in bovine endometrium and examined the physiological function and regulatory mechanism of BMP4 expression. The messenger RNA (mRNA) expression of BMP4 and its receptors was detected in bovine endometrium of both ipsilateral (corpus luteum [CL]-side) and contralateral (non-CL-side) uterine horns during the estrous cycle and early pregnancy. BMP4 protein levels were higher in the endometrial tissues obtained from those cows in early pregnancy than in the estrous cycle. Immunohistochemical analysis showed that BMP4 and its receptors were localized in endometrial epithelial cells. The addition of BMP4 to cultured endometrial epithelial cells did not affect caspase-3/-8 mRNA expression, whereas it significantly inhibited cell proliferation. Both prostaglandin (PG) E2 and PGF2α concentrations in the culture supernatant were decreased when stimulated by BMP4. Furthermore, BMP4 mRNA expression was increased by stimulation with tumor necrosis factor-α (TNF) and interferon-γ (IFNG). In conclusion, BMP4 is produced in bovine endometrial epithelial cells and may contribute to the regulation of cell proliferation and suppression of PG secretion through autocrine or paracrine mechanisms. BMP4 expression in the bovine endometrium may be regulated by TNF and IFNG.

Surgical safety and efficacy of a multi-stent system, the comet stent, in Baerveldt glaucoma implant surgery: a prospective pilot study.

PURPOSE: This study aimed to introduce a new technique for lowering intraocular pressure (IOP) using a multi-stent system after the implantation of a Baerveldt glaucoma implant (BGI) and evaluate its clinical effectiveness. STUDY DESIGN: Prospective case series. METHODS: Six patients with uncontrolled refractory glaucoma were enrolled between December 2021 and May 2022. Six 6-0 nylon sutures were preoperatively inserted into the tube of a BGI. These sutures were named "comet stents" (CSs). BGI implantation was performed, and the CSs were removed one-by-one whenever the IOP rose during the follow-up period. IOP was measured 30-60 min after the removal of each CS, and the reduction in IOP was recorded to assess the effect of CS removal. IOP reduction and the effect of CS removal on IOP reduction were evaluated for 6 months. The cut and trimmed stented tubes were examined with scanning electron microscopy, and the ratio of the patent cross-sectional area to the total luminal area (PCSA, %) and the luminal area occupation rate per stent (%) were calculated. RESULTS: The mean (±standard deviation) IOP decreased from 31.5 ± 2.8 mmHg at the baseline to 14.8 ± 8.3 mmHg at 1 month, 8.8 ± 4.7 mmHg at 3 months, and 9.2 ± 3.4 mmHg at 6 months. The IOP reduction induced by CS removal ranged from 0 to 19 mmHg. The mean PCSA was 52.7 ± 1.7%, and the mean luminal area occupation rate per stent was 7.9 ± 0.3%. CONCLUSION: The use of CSs is an effective technique for controlling IOP in a step-by-step manner after BGI surgery.

Randomized Multicenter Clinical Trial Comparing 0.1% Brimonidine/0.5% Timolol Versus 1% Dorzolamide/0.5% Timolol as Adjuncts to Prostaglandin Analogues: Aibeta Crossover Study.

INTRODUCTION: This multicenter, randomized, comparative, and investigator-masked crossover clinical trial sought to compare the efficacy and tolerability of fixed combinations of 0.1% brimonidine/0.5% timolol (BTFC) versus 1% dorzolamide/0.5% timolol (DTFC) as adjunctive therapies to prostaglandin analogues. METHODS: A total of 110 patients with open-angle glaucoma or ocular hypertension previously treated with prostaglandin analogue monotherapy were randomized to receive either BTFC or DTFC as adjunctive therapy for 8 weeks. These patients were then crossed over to the alternative treatment arm for another 8 weeks. The reduction in intraocular pressure (IOP) (primary outcome), occurrence of adverse events, ocular discomfort after instillation, and patient preference (secondary outcomes) were recorded through patient interviews. RESULTS: BTFC instillation for 8 weeks reduced IOP by 3.55 mmHg, demonstrating non-inferiority to DTFC instillation (3.60 mmHg; P < 0.0001, mixed-effects model). Although adverse events were rare with both combinations, patients reported greater discomfort with DTFC than with BTFC (P < 0.0001). More patients preferred BTFC (P < 0.0001) over DTFC, as BTFC caused minimal or no eye irritation. CONCLUSION: As BTFC offered better tolerability than DTFC with comparable reduction in IOP, we recommend it as an alternative for patients who experience ocular discomfort with DTFC-prostaglandin analogue combination therapy. TRIAL REGISTRATION NUMBER: jRCTs051190125.

Patients with glaucoma who require further reduction in intraocular pressure while undergoing monotherapy with prostaglandin analogue ophthalmic solution have been prescribed two enhanced treatment options: 0.1% brimonidine/0.5% timolol fixed combination ophthalmic solution (BTFC) and 1% dorzolamide/0.5% timolol fixed combination ophthalmic solution (DTFC). The Aibeta Crossover Study Group in Japan compared the efficacy and tolerability of fixed combinations of BTFC versus DTFC when an additional fixed combination ophthalmic solution was prescribed in patients with open-angle glaucoma or ocular hypertension who had been treated with prostaglandin analogue monotherapy. We recruited 110 patients previously treated with prostaglandin analogue monotherapy at 20 clinical centers in Japan, then randomly assigned them to two alternative treatment groups: the BTFC to DTFC group or the DTFC to BTFC group, as an adjunctive therapy to prostaglandin analogues for total of 16 weeks. We compared the reduction in intraocular pressure, occurrence of side effects, eye discomfort after instillation, and patient preference between BTFC versus DTFC instillations. The intraocular pressure reduction of BTFC instillation was comparable to that of DTFC instillation, showing non-inferiority to DTFC (3.55 mmHg vs. 3.60 mmHg; P < 0.0001, mixed-effects model). Both eye drops caused few side effects; however, patients felt greater eye discomfort with DTFC than with BTFC (P < 0.0001). Because of less eye irritation, more patients preferred BTFC (P < 0.0001) over DTFC. We can recommend using BTFC for patients who feel eye discomfort with DTFC­prostaglandin analogue combination therapy.

Differential expression of pro- and anti-angiogenic factors in the endometrium between repeat breeder and normally fertile cows.

In cattle, the establishment of appropriate endometrial vasculature during the estrous cycle is required for preparing a receptive endometrium. This study aimed to investigate 1) mRNA expression of potent pro- and anti-angiogenic factors, 2) protein localization of the anti-angiogenic factor thrombospondin (TSP), and 3) vascularity in the endometrium of repeat breeder (RB) and normally fertile (non-RB) cows. Caruncular and intercaruncular endometrium was collected from RB and non-RB cows during the luteal phase of the estrous cycle. RB cows had greater mRNA expression levels of TSP ligands (TSP1 and TSP2) and receptors (CD36 and CD47) than non-RB cows. Although the mRNA expression levels of most angiogenic factors did not change by repeat breeding, RB cows had greater mRNA expression of fibroblast growth factor receptor 1 (FGFR1), angiopoietin 1 (ANGPT1), and ANGPT2 and a less mRNA expression of vascular endothelial growth factor B (VEGFB) than non-RB cows. By immunohistochemistry, TSP1, TSP2, CD36, and CD47 were detected in the luminal epithelium, glandular epithelium, stromal cells, and blood vessels of the endometrium. Two indexes of vascularity, the number of blood vessels and the percentage of area stained positive for the von Willebrand factor, were lower in the endometrium of RB than in that of non-RB cows. These results demonstrate that RB cows have a greater expression of both ligands and receptors for the anti-angiogenic factor TSP and a reduced vascular distribution in the endometrium compared with non-RB cows, suggesting suppressed endometrial angiogenesis.

Role of CRP2-MRTF interaction in functions of myofibroblasts.

Inflammatory response induces phenotypic modulation of fibroblasts into myofibroblasts. Although transforming growth factor-ßs (TGF-ßs) evoke such transition, the details of the mechanism are still unknown. Here, we report that a LIM domain protein, cysteine-and glycine-rich protein 2 (CSRP2 [CRP2]) plays a vital role in the functional expression profile in myofibroblasts and cancer-associated fibroblasts (CAFs). Knock-down of CRP2 severely inhibits the expression of smooth muscle cell (SMC) genes, cell motility, and CAF-mediated collective invasion of epidermoid carcinoma. We elucidate the following molecular bases: CRP2 directly binds to myocardin-related transcription factors (MRTF-A/B [MRTFs]) and serum response factor (SRF) and stabilizes the MRTF/SRF/CArG-box complex to activate SMC gene expression. Furthermore, a three-dimensional structural analysis of CRP2 identifies the amino acids required for the CRP2-MRTF-A interaction. Polar amino acids in the C-terminal half (serine-152, glutamate-154, serine-155, threonine-156, threonine-157, and threonine-159 in human CRP2) are responsible for direct binding to MRTF-A. On the other hand, hydrophobic amino acids outside the consensus sequence of the LIM domain (tryptophan-139, phenylalanine-144, leucine-153, and leucine-158 in human CRP2) play a role in stabilizing the unique structure of the LIM domain.Key words: CRP2, 3D structure, myocardin-related transcription factor, myofibroblast, cancer-associated fibroblasts.

Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay.

Actin-related protein 5 (ARP5) inhibits the differentiation of skeletal, smooth, and cardiac muscle tissues, and ARP5 expression increases or decreases according to physiological and pathological changes in the muscle differentiation status. However, the regulatory mechanisms of ARP5 expression are largely unknown. Here, we identified a novel Arp5 mRNA isoform that contains premature termination codons in alternative exon 7b and is thus targeted by nonsense-mediated mRNA decay (NMD). In mouse skeletal muscle cells, switching from the canonical Arp5 isoform, i.e., Arp5(7a), to the NMD-targeted isoform Arp5(7b) occurred during differentiation, suggesting that Arp5 expression is regulated by alternative splicing coupled to NMD (AS-NMD). We developed an original method to accurately quantify the proportion of both Arp5 isoforms and measured higher levels of Arp5(7b) in muscle and brain tissues, where ARP5 is less expressed. The 3' splice site in Arp5 exon 7 has an unusual acceptor sequence that often leads to the skip of the authentic splice site and the use of the cryptic splice site localized 16 bases downstream. When the unusual acceptor sequence was mutated to the usual one, the Arp5(7b) isoform was barely detectable. The expression of several splicing factors involved in 3' splice site recognition was reduced after muscle differentiation. Additionally, knockdown of splicing factors increased the levels of Arp5(7b) and decreased the expression of Arp5(7a). Furthermore, strong positive correlations were found between Arp5 expression and the levels of these splicing factors in human skeletal and cardiac muscle tissues. Thus, Arp5 expression in muscle tissues is most likely regulated by the AS-NMD pathway.

Prevalence and characteristics of oblique astigmatism.

OBJECTIVE: To examine the incidence and characteristics of eyes with oblique astigmatism stratified by meridian, age, sex, and eye side (left to right). METHODS: One thousand eyes of 1000 patients with oblique corneal astigmatism underwent videokeratographic examination and was classified into 4 meridian categories: (1) 31°-45°, (2) 46°-59°, (3) 121°-135°, and (4) 136°-149°. Amounts of regular and irregular astigmatism, and the vertical/horizontal (Rx) and oblique astigmatism components (Ry) decomposed using vector analysis were compared among the 4 categories and age groups, and between sexes and eye sides. RESULTS: Incidences of the 4 meridian categories were similar and did not differ significantly among age groups or between sexes. The incidence was significantly greater in eyes in meridian categories 1 and 2 in the left eye and categories 3 and 4 in the right eye, and significantly greater in men in their 40 s and 50 s and in women in their 70 s and 80 s (P < 0.0001). The mean regular astigmatism, asymmetry and higher-order irregularity components, and Rx and absolute Ry significantly increased with age (P ≤ 0.0372). The mean regular and irregular astigmatism, and absolute Rx and Ry did not differ significantly among the 4 categories, or between sexes or left and right eyes. CONCLUSIONS: The incidence of oblique astigmatism was significantly greater in the temporal side meridians, and the incidence in women increased with age. The degree of oblique astigmatism increased with age, with an increase in irregular astigmatism.

Actin-related protein 5 suppresses the cooperative activation of cardiac gene transcription by myocardin and MEF2.

MYOCD is a transcription factor important for cardiac and smooth muscle development. We previously identified that actin-related protein 5 (ARP5) binds to the N-terminus of MYOCD. Here, we demonstrate that ARP5 inhibits the cooperative action of the cardiac-specific isoform of MYOCD with MEF2. ARP5 overexpression in murine hearts induced cardiac hypertrophy and fibrosis, whereas ARP5 knockdown in P19CL6 cells significantly increased cardiac gene expression. ARP5 was found to bind to a MEF2-binding motif of cardiac MYOCD and inhibit MEF2-mediated transactivation by MYOCD. RNA-seq analysis revealed 849 genes that are upregulated by MYOCD-MEF2 and 650 genes that are repressed by ARP5. ARP5 expression increased with cardiomyopathy and was negatively correlated with the expression of Tnnt2 and Ttn, which were regulated by cardiac MYOCD-MEF2. Overall, our data suggest that ARP5 is a potential suppressor of cardiac MYOCD during physiological and pathological processes.

Inflammatory Cells and Lipid Deposits Detected by in Vivo Confocal Microscopy in Brimonidine Tartrate Ophthalmic Solution-Related Corneal Disorders: A Case Series.

We reviewed the medical charts of five patients diagnosed with brimonidine tartrate (BT)-related corneal disorders. A fan-shaped corneal opacity was present in four patients and limbal corneal infiltrations were present in one patient. In vivo confocal microscopy revealed dendritic cells and lipid deposits in the fan-shaped opacity as well as neutrophils in limbal infiltrations. BT instillation was discontinued and topical administration of a corticosteroid was initiated for all patients. The limbal infiltrations improved after BT discontinuation. Conversely, the fan-shaped opacity remained in all affected patients. After a fan-shaped opacity has developed in a patient with a BT-related corneal disorder, the lesion is difficult to resolve. However, limbal infiltrations respond well to treatment. Therefore, BT should be discontinued and anti-inflammatory treatment initiated before a fan-shaped opacity forms.