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.

Chemical inhibition of the auxin inactivation pathway uncovers the roles of metabolic turnover in auxin homeostasis.

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Radicle growth regulation of root parasitic plants by auxin-related compounds.

Root parasitic plants in the Orobancheceae, such as Striga and Orobanche, cause significant damage to crop production. The germination step of these root parasitic plants is induced by host-root-derived strigolactones (SLs). After germination, the radicles elongate toward the host and invade the host root. We have previously discovered that a simple amino acid, tryptophan (Trp), as well as its metabolite, the plant hormone indole-3-acetic acid (IAA), can inhibit radicle elongation of Orobanche minor. These results suggest that auxin plays a crucial role in the radicle elongation step in root parasitic plants. In this report, we used various auxin chemical probes to dissect the auxin function in the radicle growth of O. minor and Striga hermonthica. We found that synthetic auxins inhibited radicle elongation. In addition, auxin receptor antagonist, auxinole, rescued the inhibition of radicle growth by exogenous IAA. Moreover, a polar transport inhibitor of auxin, N-1-naphthylphthalamic acid (NPA), affected radicle bending. We also proved that exogenously applied Trp is converted into IAA in O. minor seeds, and auxinole partly rescued this radicle elongation. Our data demonstrate a pivotal role of auxin in radicle growth. Thus, manipulation of auxin function in root parasitic plants should offer a useful approach to combat these parasites.

Arinole, a novel auxin-stimulating benzoxazole, affects root growth and promotes adventitious root formation.

The triple response phenotype is characteristic for seedlings treated with the phytohormone ethylene or its direct precursor 1-aminocyclopropane-carboxylic acid and is often employed to find novel chemical tools to probe ethylene responses. We identified a benzoxazole-urea derivative (B2) partially mimicking ethylene effects in a triple response bioassay. A thorough phenotypic analysis demonstrated that B2 and its closest analogue arinole (ARI) induced phenotypic responses reminiscent of seedlings with elevated levels of auxin, including impaired hook development and inhibition of seedling growth. Specifically, ARI reduced longitudinal cell elongation in roots, while promoting cell division. In contrast to other natural or synthetic auxins, ARI mostly acts as an inducer of adventitious root development, with only limited effects on lateral root development. Quantification of free auxins and auxin biosynthetic precursors as well as auxin-related gene expression demonstrated that ARI boosts global auxin levels. In addition, analyses of auxin reporter lines and mutants, besides pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment resulted in AR formation in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, a desirable trait in both agriculture and horticulture.

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.

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.

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.

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.

Prospective comparison of accuracy of intraocular lens calculation formulas in phacovitrectomy: a pilot study in a real-world clinical practice.

PURPOSE: To compare the accuracy of intraocular lens (IOL) power calculations among IOL formulas after phacovitrectomy. METHODS: We prospectively enrolled 206 eyes of 206 patients who underwent 25-gauge phacovitrectomy, without gas tamponade, for macular pathology. Pre-operative optical biometry used the IOLMaster 700 to calculate the IOL power with the new formulas, i.e. the Barrett Universal II (BU II), Emmetropia Verifying Optical version 2.0, Hill-Radial Basis Function (RBF) version 3.0, Kane, and Ladas Super Formula, and conventional formulas, i.e. Haigis, Hoffer Q, Holladay 1, Holladay 2, and Sanders-Retzlaff-Kraff/T (SRK/T). A single-piece foldable IOL was implanted in all cases. Manifest refractions were measured before and 3 months after surgery. RESULTS: The BU II formula showed the lowest standard deviation and mean and median absolute errors and had the highest percentage of eyes with a refractive prediction error within ± 0.25 D. The absolute error was significantly lower with the four new formulas, except the Hill-RBF, than with the Hoffer Q (all p = ≤ 0.010) and Holladay 1 formulas (all p = < 0.010). The absolute error with the BU II formula was also lower than that with the Holladay 2 (p = 0.012) and SRK/T (p = 0.024) formulas. CONCLUSION: Overall, the new IOL formulas, except the Hill-RBF, were superior to some of the conventional formulas for calculating IOL power in phacovitrectomy.

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.

The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants.

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition.

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.

Association of the CYP39A1 G204E Genetic Variant with Increased Risk of Glaucoma and Blindness in Patients with Exfoliation Syndrome.

PURPOSE: Carriers of functionally deficient mutations in the CYP39A1 gene have been recently reported to have a 2-fold increased risk of exfoliation syndrome (XFS). The aim of this study was to evaluate the risk of blindness and related clinical phenotypes of XFS patients carrying the loss-of-function CYP39A1 G204E mutation in comparison with XFS patients without any CYP39A1 mutation. DESIGN: Retrospective case study. PARTICIPANTS: A total of 35 patients diagnosed with XFS carrying the CYP39A1 G204E mutation and 150 XFS patients without any CYP39A1 mutation who were randomly selected from the Japanese XFS cohort. METHODS: Two-sided Fisher exact test with an alpha level < 0.05 was used to estimate the significance of the calculated odds ratio (OR) for all categorical measures. Comparisons between groups of subjects were performed using linear mixed effect models with group as random effect and taking possible dependence between eyes within a subject into account. MAIN OUTCOME MEASURES: Primary analysis compared the incidence of blindness (defined as visual acuity [VA] < 0.05 decimal), prevalence of exfoliation glaucoma (XFG), history of glaucoma surgery, and indices of glaucoma severity such as visual field (VF) mean deviation (MD), intraocular pressure (IOP), and vertical cup-disc ratio (CDR) between CYP39A1 G204E carriers and those without any CYP39A1 mutation. RESULTS: The overall risk for blindness was significantly higher in XFS patients carrying the CYP39A1 G204E variant (10/35 [28.6%]) compared with XFS patients without any CYP39A1 mutations (8/150 [5.4%]; odds ratio [OR], 7.1; 95% confidence interval [CI], 2.7-20.2]; P < 0.001). A higher proportion of XFS patients with the CYP39A1 G204E mutation (23/35 [65.7%]) had evidence of XFG in at least 1 eye compared with the comparison group (41/150 [27.3%]; OR, 5.1; 95% CI, 2.4-11.4]; P < 0.0001). Significantly higher peak IOP, larger vertical CDR, and worse VF MD were also found in CYP39A1 G204E variant carriers (P < 0.001). Additionally, patients with the CYP39A1 G204E mutation (18/35 [51.4%]) required more laser or glaucoma surgical interventions compared with those without any CYP39A1 mutation (32/150 [21.3%], P < 0.001). CONCLUSIONS: Patients with XFS carrying the CYP39A1 G204E mutation had significantly increased risk of blindness, higher occurrence of XFG, and more severe glaucoma compared with patients with XFS without any CYP39A1 mutation.

A luciferase reporter assay for ecdysone agonists using HEK293T cells.

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators.

Long-term changes in the refractive effect of a toric intraocular lens on astigmatism correction.

PURPOSE: To examine the long-term changes in the astigmatism-correcting effect of a toric intraocular lens (IOL) after stabilization of surgically induced astigmatic changes due to cataract surgery. METHODS: Unilateral eyes of 120 patients that received a toric IOL for against-the-rule (ATR) or with-the-rule (WTR) astigmatism were enrolled. Manifest refractive and anterior corneal astigmatism, and ocular residual astigmatism which is mainly derived from internal optics were examined preoperatively, at approximately 2 months postoperatively (baseline) and at 5 ~ 10 years postbaseline. The astigmatism was decomposed to vertical/horizontal (Rx) and oblique components (Ry), which was compared between baseline and 5 ~ 10 years postbaseline. RESULTS: In the eyes having ATR astigmatism, the mean Rx and Ry of the manifest refractive and corneal astigmatism significantly changed toward ATR astigmatism between the baseline and 5 ~ 10 years postbaseline (p ≤ 0.0304), but those of ocular residual astigmatism did not change significantly between the 2 time points. In the eyes having WTR astigmatism, the Rx and Ry of refractive, corneal, and ocular residual astigmatism did not change significantly between the 2 time points. Double-angle plots revealed an ATR shift in refractive and corneal astigmatism and no marked change in the ocular residual astigmatism in the eyes with ATR astigmatism, and there is no change in this astigmatism in the eyes with WTR astigmatism. CONCLUSION: The long-term changes with age in the effect of a toric IOL significantly deteriorated due to an ATR shift of corneal astigmatism in the eyes having ATR astigmatism, while it was maintained in eyes having WTR astigmatism, suggesting that ATR astigmatism should be overcorrected.

Direct effects of linoleic and linolenic acids on bovine uterine function using in vivo and in vitro studies.

The aim of the present study was to evaluate the effects of continuous administration of linoleic acid or linolenic acid into the intra-uterine horn, ipsilateral to the corpus luteum, on the duration of the estrous cycle and plasma progesterone (P4) concentration. The effects of linoleic and linolenic acids on bovine uterine and luteal functions were also studied using a tissue culture system. Intra-uterine administration of linoleic or linolenic acid (5 mg/10 ml of each per day) in cows, between days 12 and 21, resulted in a prolonged estrous cycle compared to the average duration of the last one to three estrous cycles before administration in each group (P < 0.05). Moreover, plasma P4 concentration in cows treated with linoleic or linolenic acid was high between days 19 and 21 (linoleic acid), or on day 20 (linolenic acid), compared to that of the control cows (saline administration; P < 0.05 or lower). Both linoleic (500 µg/ml) and linolenic (5 and 500 µg/ml) acids stimulated prostaglandin (PG) E2 but inhibited PGF2α production by cultured endometrial tissue (P < 0.01), while P4 production by cultured luteal tissue was not affected. These findings suggest that both linoleic and linolenic acids support luteal P4 production by regulating endometrial PG production and, subsequently, prolonging the duration of the estrous cycle in cows.

Fluorescent Auxin Analogs Report Two Auxin Binding Sites with Different Subcellular Distribution and Affinities: A Cue for Non-Transcriptional Auxin Signaling.

The complexity of auxin signaling is partially due to multiple auxin receptors that trigger differential signaling. To obtain insight into the subcellular localization of auxin-binding sites, we used fluorescent auxin analogs that can undergo transport but do not deploy auxin signaling. Using fluorescent probes for different subcellular compartments, we can show that the fluorescent analog of 1-naphthaleneacetic acid (NAA) associates with the endoplasmic reticulum (ER) and tonoplast, while the fluorescent analog of indole acetic acid (IAA) binds to the ER. The binding of the fluorescent NAA analog to the ER can be outcompeted by unlabeled NAA, which allows us to estimate the affinity of NAA for this binding site to be around 1 µM. The non-transportable auxin 2,4-dichlorophenoxyacetic acid (2,4-D) interferes with the binding site for the fluorescent NAA analog at the tonoplast but not with the binding site for the fluorescent IAA analog at the ER. We integrate these data into a working model, where the tonoplast hosts a binding site with a high affinity for 2,4-D, while the ER hosts a binding site with high affinity for NAA. Thus, the differential subcellular localization of binding sites reflects the differential signaling in response to these artificial auxins.

Case Reports on Effect of Intratympanic Steroid Injection Therapy on Late-Onset Hearing Loss in Congenital Cytomegalovirus Infection.

BACKGROUND: One of the conditions or symptoms caused by congenital cytomegalovirus (cCMV) infection is late-onset hearing loss. This report examines the cases of two children exhibiting late-onset hearing loss after cCMV infection who showed improvement in hearing after undergoing intratympanic steroid injection therapy (IST). CASES: Case1 is girl aged 8 years and 10 months and case2 is girl aged 5 years and 1 month. Cytomegalovirus (CMV) was not detected in the blood or urine of either child at the time of hearing loss despite them having cCMV infection. FINDINGS: The hearing of both children improved as a result of IST on an outpatient basis. Case1 was given first session of IST in left ear immediately on the day of her visit and second session of IST in left ear 2 days later. Tendency for improvement in threshold on left side was observed (the differences were about 20 to 45 dB). Case2 was given a total of 2 sessions of IST on left ear, 3 days and 5 days after visiting the hospital. The test result of distortion product otoacoustic emissions changed from refer to pass. Tendency for improvement in threshold on left side was observed (the differences were about 5 to 25 dB). CONCLUSIONS: Bearing in mind that late-onset hearing loss in patients with cCMV may be caused by other factors besides CMV, consideration of IST as a possible treatment option is proposed.