Measuring Vision With Temporally Modulated Stripes in Infants and Children with ROP

Purpose. To determine differences in preferential looking (PL) acuities using stationary and temporally modulated stripe patterns in patients with various stages of retinopathy of prematurity (ROP). Methods. We measured the PL acuities of 134 patients (ages 4 mo to 13 yr) with various stages of ROP. Patients were divided into six subgroups according to PL vision measured with stationary stripes: (1) equal to or better than 20/200 (n = 24); (2) worse than 20/200 to 20/400 (n = 10); (3) worse than 20/400 to 20/800 (n = 15); (4) worse than 20/800 to 20/1600 (n = 13); (5) worse than 20/1600 to 20/6400 (n = 26); and (6) worse than 20/6400 (n = 46; no stationary vision). Results. In the group with PL acuity equal to or better than 20/200, no difference in vision was apparent between the two methods. In patients with acuities worse than 20/200 to 20/400, the temporally modulated PL acuities were 0.23 octave better than the PL acuities measured with the stationary stripes. The difference increased to 0.86 and 1.12 octaves in the groups with visual acuities worse than 20/400 to 20/800 and worse than 20/800 to 20/1600, respectively. The difference in the group with PL acuities worse than 20/1600 to 20/6400 was 1.69 octaves. The 46 patients with no stationary vision detected only the temporally modulated stripes. Conclusions. The results suggest that the PL acuity difference between the temporally modulated and stationary stripes increases with visual impairment. Measuring PL acuity with temporally modulated stripes is an important addition to the evaluation of severely visually impaired subjects. Invest Ophthalmol Vis Sci. 1993;34:496-502. A he preferential looking (PL) test has been useful in clinical settings to evaluate vision in infants and young children. However, some patients with severe visual impairment cannot discriminate the lowest spatial freFrom. th

Identification of androgen receptor phosphorylation in the primate ovary in vivo

The androgen receptor (AR) is a member of the nuclear receptor superfamily, and is important for both male and female reproductive health. The receptor is a target for a number of post-translational modifications including phosphorylation, which has been intensively studied in vitro. However, little is known about the phosphorylation status of the receptor in target tissues in vivo. The common marmoset is a useful model for studying human reproductive functions, and comparison of the AR primary sequence from this primate shows high conservation of serines known to be phosphorylated in the human receptor and corresponding flanking amino acids. We have used a panel of phosphospecific antibodies to study AR phosphorylation in the marmoset ovary throughout the follicular phase and after treatment with GNRH antagonist or testosterone propionate. In normal follicular phase ovaries, total AR (both phosphorylated and non-phosphorylated forms) immunopositive staining was observed in several cell types including granulosa cells of developing follicles, theca cells and endothelial cells lining blood vessels. Receptor phosphorylation at serines 81, 308, and 650 was detected primarily in the granulosa cells of developing follicles, surface epithelium, and vessel endothelial cells. Testosterone treatment lead to a modest increase in AR staining in all stages of follicle studied, while GNRH antagonist had no effect. Neither treatment significantly altered the pattern of phosphorylation compared to the control group. These results demonstrate that phosphorylation of the AR occurs, at a subset of serine residues, in a reproductive target tissue in vivo, which appears refractory to hormonal manipulations

Modelling and prevention of acute kidney injury through ischemia and reperfusion in a combined human renal proximal Tubule/Blood Vessel-on-a-Chip

Analytical BioScience

Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD

The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions

EWS/FLI Mediates Transcriptional Repression via NKX2.2 during Oncogenic Transformation in Ewing's Sarcoma

EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip) revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease

Transport characteristics of guanidino compounds at the blood-brain barrier and blood-cerebrospinal fluid barrier: relevance to neural disorders

Guanidino compounds (GCs), such as creatine, phosphocreatine, guanidinoacetic acid, creatinine, methylguanidine, guanidinosuccinic acid, γ-guanidinobutyric acid, β-guanidinopropionic acid, guanidinoethane sulfonic acid and α-guanidinoglutaric acid, are present in the mammalian brain. Although creatine and phosphocreatine play important roles in energy homeostasis in the brain, accumulation of GCs may induce epileptic discharges and convulsions. This review focuses on how physiologically important and/or neurotoxic GCs are distributed in the brain under physiological and pathological conditions. Transporters for GCs at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB) have emerged as substantial contributors to GCs distribution in the brain. Creatine transporter (CRT/solute carrier (SLC) 6A8) expressed at the BBB regulates creatine concentration in the brain, and represents a major pathway for supply of creatine from the circulating blood to the brain. CRT may be a key factor facilitating blood-to-brain guanidinoacetate transport in patients deficient in S-adenosylmethionine:guanidinoacetate N-methyltransferase, the creatine biosynthetic enzyme, resulting in cerebral accumulation of guanidinoacetate. CRT, taurine transporter (TauT/SLC6A6) and organic cation transporter (OCT3/SLC22A3) expressed at the BCSFB are involved in guanidinoacetic acid or creatinine efflux transport from CSF. Interestingly, BBB efflux transport of GCs, including guanidinoacetate and creatinine, is negligible, though the BBB has a variety of efflux transport systems for synthetic precursors of GCs, such as amino acids and neurotransmitters. Instead, the BCSFB functions as a major cerebral clearance system for GCs. In conclusion, transport of GCs at the BBB and BCSFB appears to be the key determinant of the cerebral levels of GCs, and changes in the transport characteristics may cause the abnormal distribution of GCs in the brain seen in patients with certain neurological disorders

Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression

<p>Abstract</p> <p>Background</p> <p><it>Drosophila </it>Groucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLE's modify Wnt signaling is by direct competition with β-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.</p> <p>Methods</p> <p>We used <it>in vitro </it>protein interaction assays with the LEF/TCF family member LEF-1, and <it>in vivo </it>assays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.</p> <p>Results</p> <p>Mapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay. <it>Drosophila </it>Groucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1•β-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated β-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.</p> <p>Conclusion</p> <p>Our data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with β-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.</p

Cyclooxygenase inhibitors impair CD4 T cell immunity and exacerbate Mycobacterium tuberculosis infection in aerosol-challenged mice

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), kills over 1.6 million people each year despite availability of antibiotics. The increase in drug resistant Mtb strains is a major public health emergency and host-directed therapy as adjunct to antibiotic treatment has gained increased interest. Cyclooxygenase inhibitors (COXi) are frequently used drugs to alleviate tuberculosis related symptoms. Mouse studies of acute intravenous Mtb infection have suggested a potential benefit of COXi for host-directed therapy. Here we show that COXi treatment (ibuprofen and celecoxib) is detrimental to Mtb control in different mouse models of respiratory infection. This effect links to impairments of the Type-1 helper (Th1) T-cell response as CD4 T-cells in COXi-treated animals have significantly decreased Th1 differentiation, reduced IFNγ expression and decreased protective capacity upon adoptive transfer. If confirmed in clinical trials, these findings could have major impact on global health and question the use of COXi for host-directed therapy.publishedVersio

Tim3 binding to galectin-9 stimulates antimicrobial immunity

The interaction between Tim3 on Th1 cells and galectin-9 on Mycobacterium tuberculosis–infected macrophages restricts the bacterial growth by stimulating caspase-1–dependent IL-1β secretion