Spread of activation and interaction between channels with multi-channel optogenetic stimulation in the mouse cochlea.

For individuals with severe to profound hearing loss resulting from irreversibly damaged hair cells, cochlear implants can be used to restore hearing by delivering electrical stimulation directly to the spiral ganglion neurons. However, current spread lowers the spatial resolution of neural activation. Since light can be easily confined, optogenetics is a technique that has the potential to improve the precision of neural activation, whereby visible light is used to stimulate neurons that are modified with light-sensitive opsins. This study compares the spread of neural activity across the inferior colliculus of the auditory midbrain during electrical and optical stimulation in the cochlea of acutely deafened mice with opsin-modified spiral ganglion neurons (H134R variant of the channelrhodopsin-2). Monopolar electrical stimulation was delivered via each of four 0.2 mm wide platinum electrode rings at 0.6 mm centre-to-centre spacing, whereas 453 nm wavelength light was delivered via each of five 0.22 × 0.27 mm micro-light emitting diodes (LEDs) at 0.52 mm centre-to-centre spacing. Channel interactions were also quantified by threshold changes during simultaneous stimulation by pairs of electrodes or micro-LEDs at different distances between the electrodes (0.6, 1.2 and 1.8 mm) or micro-LEDs (0.52, 1.04, 1.56 and 2.08 mm). The spread of activation resulting from single channel optical stimulation was approximately half that of monopolar electrical stimulation as measured at two levels of discrimination above threshold (p<0.001), whereas there was no significant difference between optical stimulation in opsin-modified deafened mice and pure tone acoustic stimulation in normal-hearing mice. During simultaneous micro-LED stimulation, there were minimal channel interactions for all micro-LED spacings tested. For neighbouring micro-LEDs/electrodes, the relative influence on threshold was 13-fold less for optical stimulation compared electrical stimulation (p<0.05). The outcomes of this study show that the higher spatial precision of optogenetic stimulation results in reduced channel interaction compared to electrical stimulation, which could increase the number of independent channels in a cochlear implant. Increased spatial resolution and the ability to activate more than one channel simultaneously could lead to better speech perception in cochlear implant recipients.

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry.

The screening of covalent or 'reactive' fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

Transmural impedance detects graded changes of inflammation in experimental colitis.

Ulcerative colitis is a chronic disease in which the mucosa of the colon or rectum becomes inflamed. An objective biomarker of inflammation will provide quantitative measures to support qualitative assessment during an endoscopic examination. Previous studies show that transmural electrical impedance is a quantifiable biomarker of inflammation. Here, we hypothesize that impedance detects spatially restricted areas of inflammation, thereby allowing the distinction between regions that differ in their severity of inflammation. A platinum ball electrode was placed into minimally inflamed (i.e. normal) or 2,4,6-trinitrobenzene sulphonic acid (TNBS)-inflamed colonic regions of rats and impedance measurements obtained by passing current between the intraluminal and subcutaneous return electrode. Histology of the colon was correlated with impedance measurements. The impedance of minimally inflamed (normal) tissue was 1.5-1.9 kΩ. Following TNBS injection, impedance significantly decreased within the inflammatory penumbra (p < 0.05), and decreased more in the inflammatory epicentre (p = 0.02). Histological damage correlated with impedance values (p < 0.05). Thus, impedance values of 1.5-1.9, 1.3-1.4 and 0.9-1.1 kΩ corresponded to minimally inflamed, mildly inflamed and moderately inflamed tissue, respectively. In conclusion, transmural impedance is an objective, spatially localized biomarker of mucosal integrity, and distinguishes between severities of intestinal inflammation.

Mimicking salmochelin S1 and the interactions of its Fe(III) complex with periplasmic iron siderophore binding proteins CeuE and VctP.

A mimic of the tetradentate stealth siderophore salmochelin S1, was synthesised, characterised and shown to form Fe(III) complexes with ligand-to-metal ratios of 1:1 and 3:2. Circular dichroism spectroscopy confirmed that the periplasmic binding proteins CeuE and VctP of Campylobacter jejuni and Vibrio cholerae, respectively, bind the Fe(III) complex of the salmochelin mimic by preferentially selecting Λ-configured Fe(III) complexes. Intrinsic fluorescence quenching studies revealed that VctP binds Fe(III) complexes of the mimic and structurally-related catecholate ligands, such as enterobactin, bis(2, 3-dihydroxybenzoyl-l-serine) and bis(2, 3-dihydroxybenzoyl)-1, 5-pentanediamine with higher affinity than does CeuE. Both CeuE and VctP display a clear preference for the tetradentate bis(catecholates) over the tris(catecholate) siderophore enterobactin. These findings are consistent with reports that V. cholerae and C. jejuni utilise the enterobactin hydrolysis product bis(2, 3-dihydroxybenzoyl)-O-seryl serine for the acquisition of Fe(III) and suggest that the role of salmochelin S1 in the iron uptake of enteric pathogens merits further investigation.

Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4- siderophore analogues of varied linker length.

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4-) to five, six and eight (5-, 6-, 8-LICAM4-, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4- structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4- is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4- but decreases for 6- and 8-LICAM4-. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.

Co-exposure to sunlight enhances the toxicity of naturally weathered Deepwater Horizon oil to early lifestage red drum (Sciaenops ocellatus) and speckled seatrout (Cynoscion nebulosus).

The 2010 Deepwater Horizon oil spill resulted in the accidental release of millions of barrels of crude oil into the Gulf of Mexico. Photo-induced toxicity following co-exposure to ultraviolet (UV) radiation is 1 mechanism by which polycyclic aromatic hydrocarbons (PAHs) from oil spills may exert toxicity. Red drum and speckled seatrout are both important fishery resources in the Gulf of Mexico. They spawn near-shore and produce positively buoyant embryos that hatch into larvae in approximately 24 h. The goal of the present study was to determine whether exposure to UV as natural sunlight enhances the toxicity of crude oil to early lifestage red drum and speckled seatrout. Larval fish were exposed to several dilutions of high-energy water-accommodated fractions (HEWAFs) from 2 different oils collected in the field under chain of custody during the 2010 spill and 3 gradations of natural sunlight in a factorial design. Co-exposure to natural sunlight and oil significantly reduced larval survival compared with exposure to oil alone. Although both species were sensitive at PAH concentrations reported during the Deepwater Horizon spill, speckled seatrout demonstrated a greater sensitivity to photo-induced toxicity than red drum. These data demonstrate that even advanced weathering of slicks does not ameliorate the potential for photo-induced toxicity of oil to these species. Environ Toxicol Chem 2017;36:780-785. © 2016 SETAC.

APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer.

Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

Co-regulated gene expression by oestrogen receptor α and liver receptor homolog-1 is a feature of the oestrogen response in breast cancer cells.

Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1-regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα-regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.

Elevated international normalized ratio values associated with concomitant use of warfarin and ceftriaxone.

PURPOSE: The case of a patient receiving long-term warfarin therapy who experienced elevated International Normalized Ratio (INR) values on two occasions after injections of ceftriaxone is reported. SUMMARY: An elderly woman (age, 67 years) with multiple comorbidities who had been receiving warfarin therapy for about 8 years was given an intramuscular injection of ceftriaxone 1 g for the treatment of a urinary tract infection. Four days later, her INR (which had recently ranged from 1.9 to 3.0 at a weekly warfarin dosage of 52.5-54.5 mg) was 10.74. One scheduled warfarin dose was withheld and 5 mg of phytonadione administered; one day later, the INR was 3.4 (goal, 2.5-3.5). INR values remained stable for several weeks until the patient again received a 1-g ceftriaxone injection for an infection (she was also prescribed oral cefuroxime and phenazopyridine); four days later, the INR was 16.99. Again, the scheduled warfarin dose was withheld and 5 mg of phytonadione administered. One day later, the INR had declined to 4.6 but remained above the target range; therefore, warfarin was withheld for a second day, after which the patient received 7.5 mg of warfarin sodium daily for two days, resulting in an INR decrease to 2.1. The patient continued to receive 7.5 mg of warfarin sodium daily, and at one-week follow-up the INR value (2.5) was within the therapeutic range. CONCLUSION: A 67-year-old American Indian woman with previously stable INR values during long-term warfarin therapy after mitral valve replacement surgery had INR elevations on two occasions after receiving ceftriaxone for urinary tract infections.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth.

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells.

Estrogen receptor-α (ER) is expressed in the great majority of breast cancers, and the inhibition of ER action is a key part of breast cancer treatment. The inhibition of ER action is achieved using anti-estrogens, primarily tamoxifen, and with aromatase inhibitors that inhibit estrogen biosynthesis, thereby preventing ER activation. However, resistance to these therapies is common. With the aim of identifying new molecular targets for breast cancer therapy, we have identified the liver receptor homolog-1 (LRH-1) as an estrogen-regulated gene. RNA interference and over-expression studies were used to investigate the role of the LRH-1 in regulating breast cancer growth and to identify the targets of an LRH-1 action. Promoter recruitment was determined using reporter gene and chromatin immunoprecipitation (ChIP) assays. We show that LRH-1 regulates breast cancer cell growth by regulating the ER expression. Reporter gene and in vitro DNA-binding assays identified an LRH-1-binding site in the ER gene promoter, and ChIP assays have demonstrated in vivo binding at this site. We also provide evidence for new LRH-1 variants in breast cancer cells arising from the use of alternative promoters. Previous studies have shown that LRH-1 functions in estrogen biosynthesis by regulating aromatase expression. Our findings extend this by highlighting LRH-1 as a key regulator of the estrogen response in breast cancer cells through the regulation of ER expression. Hence, inhibition of LRH-1 could provide a powerful new approach for the treatment of endocrine-resistant breast cancer.

Função do interferon-tau durante o reconhecimento materno da gestação em ruminantes / The role of interferon-tau during maternal recognition of pregnancy in ruminants

O reconhecimento materno da gestação é o período em que o concepto sinaliza sua presença para a mãe. Em ruminantes, este período coincide com o alongamento do embrião e a máxima produção de interferon-tau (IFNT). O IFNT produzido pelo concepto age via parácrina no útero inibindo a expressão dos receptores de estrógenos (ESR1) e de ocitocina (OXTR) no epitélio luminal do endométrio, evitando, assim, a liberação de pulsos luteolíticos de prostaglandina F2 alfa (PGF2 ), hormonio responsável pelo início da luteólise. Além da sua ação durante o reconhecimento materno da gestação em ruminantes, o IFNT aumenta a expressão de vários genes estimulados por interferons (ISGs) no útero, no corpo lúteo (CL) e em células sanguíneas. Estudos recentes demonstraram que o IFNT possui ação endócrina no CL ovino e também estende o ciclo estral (pseudo gestação) além do dia 32 após a infusão de IFNT recombinante ovino (roIFNT) na veia uterina. A comprovação da saída de IFNT do útero pela veia uterina sugere que a ação endócrina do IFNT possa ser um mecanismo complementar ao mecanismo intrauterino de reconhecimento materno da gestação. A ação direta do IFNT em tecidos extrauterinos estimula a expressão de ISGs que, no CL, podem estar envolvidos com a resistência luteal à ação luteolítica da PGF2a.

Maternal recognition of pregnancy is the period when the conceptus signals its presence to the dam. In ruminants, it requires conceptus elongation, which coincides with maximum production of interferon-tau (IFNT). Conceptus IFNT acts in a paracrine manner silencing estrogen receptor alpha (ESR1) and oxytocin receptor (OXTR) in the luminal epithelium, thus preventing luteolytic prostaglandin F2 alpha (PGF2 ) pulses. Besides its role during maternal recognition of pregnancy, IFNT induces the expression of several interferon stimulated genes (ISGs) in the endometrium, corpus luteum (CL) and blood cells. Recently, it was suggested an endocrine role for IFNT during the period of maternal recognition of pregnancy in sheep. It was demonstrated that infusion of IFNT into the uterine vein can extend the estrous cycle beyond 32 days. This direct action of IFNT in extrauterine tissues induces ISGs expression, which might be involved in the rescue of the CL from the luteolytic effects of PGF2 pulses.

CK1delta modulates the transcriptional activity of ERalpha via AIB1 in an estrogen-dependent manner and regulates ERalpha-AIB1 interactions.

Oncogenesis in breast cancer often requires the overexpression of the nuclear receptor coactivator AIB1/SRC-3 acting in conjunction with estrogen receptor-alpha (ERalpha). Phosphorylation of both ERalpha and AIB1 has been shown to have profound effects on their functions. In addition, proteasome-mediated degradation plays a major role by regulating their stability and activity. CK1delta, a member of the ubiquitous casein kinase-1 family, is implicated in the progression of breast cancer. In this study, we show that both ERalpha and AIB1 are substrates for CK1delta in vitro, and identify a novel AIB1 phosphorylation site (S601) targeted by CK1delta, significant for the co-activator function of AIB1. CK1delta is able to interact with ERalpha and AIB1 in vivo, while overexpression of CK1delta in breast cancer cells results in an increased association of ERalpha with AIB1 as confirmed by co-immunoprecipitation assays from cell lysates. Using an siRNA-based approach, luciferase reporter assays and qRT-PCR, we observe that silencing of CK1delta leads to reduced ERalpha transcriptional activity, despite increased ERalpha levels, similarly to proteasome inhibition. We provide evidence that AIB1 protein levels are reduced by CK1delta silencing, in an estradiol-dependent manner; such destabilization can be inhibited by pre-treatment with the proteasome inhibitor MG132. We propose that differing activities adopted by ERalpha and AIB1 as a consequence of their interactions with and phosphorylation by CK1delta, particularly AIB1 stabilization, influence the transcriptional activity of ERalpha, and therefore have a role in breast cancer development.

Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-alpha activity.

Phosphorylation of estrogen receptor-alpha (ERalpha) at specific residues in transcription activation function 1 (AF-1) can stimulate ERalpha activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERalpha activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERalpha activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERalpha activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERalpha activity. Collectively, these data indicate that the MAPK stimulation of ERalpha activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERalpha at these sites may contribute to resistance to tamoxifen in breast cancer.

ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases.

The regulation of gene expression by estrogen receptor-alpha (ERalpha) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERalpha as bait in a yeast two-hybrid screen, we have identified a novel ERalpha-interacting protein, ZNF366, which is a potent corepressor of ERalpha activity. The interaction between ZNF366 and ERalpha has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERalpha corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation.

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.