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In cystic fibrosis (CF), reduced HCOsub3/subsup-/supsecretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHsubASL/sub) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCOsub3/subsup-/supand Clsup-/suptransport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHsubASL/sub. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHsubASL/sub. This response was HCOsub3/subsup-/supdependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed twoiWNK/iisoforms in secretory cells and ionocytes, and knockdown of eitheriWNK1/ioriWNK2/iincreased CF pHsubASL/sub. WNK inhibition decreased Clsup-/supsecretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Clsup-/supsubstitution also alkalinized CF pHsubASL/sub. These data suggest that WNK kinases influence the balance between transepithelial Clsup-/supversus HCOsub3/subsup-/supsecretion. Moreover, reducing basolateral Clsup-/supentry may increase HCOsub3/subsup-/supsecretion and raise pHsubASL/sub, thereby improving CF host defenses.

Vertebrate replication forks arrested at interstrand DNA cross-links (ICLs) engage the Fanconi anemia pathway to incise arrested forks, 'unhooking' the ICL and forming a double strand break (DSB) that is repaired by homologous recombination (HR). The FANCP product, SLX4, in complex with the XPF (also known as FANCQ or ERCC4)-ERCC1 endonuclease, mediates ICL unhooking. Whether this mechanism operates at replication fork barriers other than ICLs is unknown. Here, we study the role of mouse SLX4 in HR triggered by a site-specific chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus-Ter complex. We show that SLX4-XPF is required for Tus-Ter-induced HR but not for error-free HR induced by a replication-independent DSB. We additionally uncover a role for SLX4-XPF in DSB-induced long-tract gene conversion, an error-prone HR pathway related to break-induced replication. Notably, Slx4 and Xpf mutants that are defective for Tus-Ter-induced HR are hypersensitive to ICLs and also to the DNA-protein cross-linking agents 5-aza-2'-deoxycytidine and zebularine. Collectively, these findings show that SLX4-XPF can process DNA-protein fork barriers for HR and that the Tus-Ter system recapitulates this process.

The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl−/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+–2HCO3− stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3− influx. However, NBCe1 in the kidney mediates HCO3− efflux (HCO3− reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3− efflux) than in murine colons (in which NBCe1 mediates HCO3− influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+–2HCO3− stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3− influx. However, NBCe1 in the kidney mediates HCO3− efflux (HCO3− reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3− efflux) than in murine colons (in which NBCe1 mediates HCO3− influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

Without cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) HCO(3)(–) secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl(–)/HCO(3)(–) exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator–induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO(3)(–) secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.

SummaryConservative repair of stalled replication forks is important for the maintenance of a stable genome. However, the mechanisms that regulate repair pathway “choice” at stalled mammalian forks remain poorly understood. The Fanconi anemia complementation group M gene, FANCM, encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here we use a chromosomally integrated reporter of stalled fork repair, in combination with defined mutations engineered within the endogenous Fancm gene in primary mammalian cells, to study how Fancm regulates stalled fork repair. We identify separation-of-function Fancm mutants, which reveal that distinct repair functions of FANCM are enacted by modular, molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, a mutation that inactivates the ATPase function of FANCM disables all FANCM-mediated repair functions and appears to “trap” FANCM at stalled forks. We find that Fancm null cells do not survive genetic inactivation of Brca1. This synthetic lethal interaction is recapitulated in Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising “druggable” target for therapy of BRCA1 mutant cancers.

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.

Purpose An adverse effect of amantadine, a drug used for Parkinson's disease, is corneal edema. While corneal endothelial cell loss is noted with amantadine toxicity, the reversibility of corneal edema suggests that amantadine affects active mechanisms regulating corneal hydration. Although mainly known as a NMDA receptor antagonist, amantadine is also a K+-channel blocker. The purpose of this study was to investigate potential mechanisms of amantadine's toxic effects on corneal endothelium. Methods Bovine corneas were used for short-circuit current measurements of corneal endothelial active ion transport to compare the effects of amantadine with an NMDA receptor agonist (NMDA) and antagonist (D-APV), and the K+-channel blockers BaCl2 and clotrimazole. Cell death and changes in cell morphology were observed using annexin V stain, alizarin red S staining of the intercellular junctions, ZO-1 immunolocalization, and phalloidin stain of the actin cytoskeleton. Results Amantadine caused a transient decrease in the short-circuit current that mimicked the effect of clotrimazole. BaCl2, and the NMDA receptor agonist and antagonist had no effect on the short-circuit current. Tissue incubation with amantadine caused an increase in cell area (measured by ZO-1 localization) and cell height (measured by phalloidin stain) but did not increase apoptotic cell death (annexin V stain). Conclusions The similarity of amantadine and clotrimazole effects on the short-circuit current and the effects on cell volume suggest that amantadine's actions on corneal endothelium are mediated via K+ channels. The observed absence of cell death and transient effect on short-circuit current support the reported reversibility of amantadine–induced corneal edema.

Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative “short tract” gene conversion (STGC) induced by a chromosomal Tus/Ter RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/Ter-induced STGC proceeds via a two-ended DSB intermediate. If so, Tus/Ter-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/Ter-induced HR is unaffected by deletion of either of two C-NHEJ genes, Xrcc4 or Ku70. These results show that Tus/Ter-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/Ter-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/Ter RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/Ter was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/Ter was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/Ter RFB are not conventional DSBs. We propose that Rad51 acts as an “early responder” at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery., Author summary Genomic instability is a significant contributor to human disease, ranging from hereditary developmental disorders to cancer predisposition. Two major triggers to genomic instability are chromosomal double strand breaks (DSBs) and the stalling of replication forks during the DNA synthesis (S phase) of the cell cycle. The “rules” that govern mammalian DSB repair are increasingly well understood, and it is recognized that the two major DSB repair pathways—classical non-homologous end joining (C-NHEJ) and homologous recombination (HR)—compete to repair a mammalian DSB. In contrast, we do not yet have equivalent insight into the regulation of repair at sites of mammalian replication fork stalling. Here, we explore the relationship between C-NHEJ and HR at a defined chromosomal replication fork barrier in mammalian cells. We show that, in contrast to DSB repair, repair at stalled forks does not entail competition between C-NHEJ and HR. We find that Rad51, a key mediator of HR, accumulates in an intense and highly localized fashion at the stalled fork. Based upon these findings, we propose a model of HR initiation at the stalled fork in which a Rad51-mediated fork remodeling step prevents access of C-NHEJ to the stalled fork.

Patients on warfarin therapy need to achieve and maintain anticoagulation control in order to experience the benefits of treatment while minimizing bleeding risk. Low health literacy skills may hinder patients' ability to use and adhere to warfarin in a safe and effective manner. The authors conducted this study to evaluate the relationship between health literacy and anticoagulation control among patients on chronic warfarin therapy. Participants were recruited from 2 diverse anticoagulation clinics in North Carolina. Time in therapeutic range (TTR) for warfarin therapy was used as a measure of anticoagulation control. Health literacy was assessed using the short form of the Test of Functional Health Literacy in Adults (S-TOFHLA). Of the 198 study participants, 51% had limited health literacy (S-TOFHLA score of 0-90) and 33% had poor anticoagulation control (TTR

Ventilation and mixing systems in the Hanford Waste Treatment and Immobilization Plant (WTP) are being designed to account for the flammable gas hydrogen that will form in process streams, just as it also does in the radioactive liquid wastes awaiting immobilization at the Hanford Tank Farms. Tank wastes forming hydrogen at the highest rates do so by reactions involving dissolved organic complexant compounds, even though hydrogen is also formed by the better known radiolysis pathway. Hydrogen generation rates (HGRs) are predicted with a correlation relating waste properties to reaction pathways involving radiolysis of water and the degradation of organic compounds. This correlation accounts only for aqueous phase reactions. An antifoam agent (AFA) will be added to waste processed in the WTP. This organic liquid mixture is immiscible in aqueous systems and will therefore form a nonaqueous phase liquid layer on the processed waste, unless some of its compounds are unstable in the hostile physical/chemical environment and break down into soluble degradation products. Dissolved organic species increase the organic source term in the WTP HGR correlation, but the correlation requires adaptation to address hydrogen formed from immiscible organic liquids. Here, we report our initial evaluation of the hydrogen formed by 60 Co gamma irradiation of a waste simulant containing Dow Corning Q2-3183A AFA with an adapted WTP HGR correlation.

KCNQ1 (Kv7.1 or KvLQT1) encodes the alpha-subunit of a voltage-gated potassium channel found in tissues including heart, brain, epithelia and smooth muscle. Tissue-specific characteristics of KCNQ1 current are diverse, due to modification by ancillary subunits. In heart, KCNQ1 associates with KCNE1 (MinK), producing a slowly activating voltage-dependent channel. In epithelia, KCNQ1 co-assembles with KCNE3 (Mirp2) producing a constitutively open channel. Chromanol 293B is a selective KCNQ1 blocker. We studied drug binding and frequency dependence of 293B on KCNQ1 and ancillary subunits expressed in Xenopus oocytes. Ancillary subunits altered 293B potency up to 100-fold (IC50 for KCNQ1 = 65.4 ± 1.7 μm; KCNQ1/KCNE1 = 15.1 ± 3.3 μm; KCNQ1/KCNE3 = 0.54 ± 0.18 μm). Block of KCNQ1 and KCNQ1/KCNE3 was time independent, but 293B altered KCNQ1/KCNE1 activation. We therefore studied frequency-dependent block of KCNQ1/KCNE1. Repetitive rapid stimulation increased KCNQ1/KCNE1 current biphasically, and 293B abolished the slow component. KCNQ1/KCNE3[V72T] activates slowly with a KCNQ1/KCNE1-like phenotype, but retains the high affinity binding of KCNQ1/KCNE3, demonstrating that subunit-mediated changes in gating can be dissociated from subunit-mediated changes in affinity. This study demonstrates the KCNQ1 pharmacology is significantly altered by ancillary subunits. The response of KCNQ1 to specific blockers will therefore be critically dependent on the electrical stimulation pattern of the target organ. Furthermore, the dissociation between gating and overall affinity suggests that mutations in ancillary subunits can potentially strongly alter drug sensitivity without obvious functional changes in gating behaviour, giving rise to unexpected side-effects such as a predisposition to acquired long QT syndrome.

Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli . Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC.

Several benchscale column tests (resin bed volume ≤75 mL) have been conducted with SuperLig® 639 resin and simulated U.S. Department of Energy tank waste supernates. Rhenium (surrogate for technetium in actual waste samples) breakthrough profiles were determined for three simulant compositions, which are representative of the basic waste categories requiring treatment in the Hanford River Protection Project Waste Treatment Plant. Considerable loading performance variability was observed between the three waste types, although the resin is effective at rhenium removal from each solution. Careful and frequent analysis during elution studies conducted at the conclusion of the column loading tests confirmed that sodium nitrate and sodium perrhenate ion pairs are primary sorbing species on SuperLig® 639 resin. Furthermore, it was discovered that potassium nitrate and potassium perrhenate salts are significant competitors for sorption sites on the resin. Successive desorption profiles were identified f...

Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5′-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5–2 μM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-( p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2breceptors. These findings provide new insight into how EPEC causes watery diarrhea.

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.

Fecal constituents such as bile acids and increased sialylation of membrane glycoproteins by alpha-2,6-sialyltransferase (HST6N-1) may contribute to colorectal tumorigenesis. We hypothesized that bile acids and phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] would upregulate HST6N-1 in colonic cells. However, deoxycholate (DOC) (300 mumol/l), a secondary bile acid, and TPA (20 ng/ml) decreased expression of an approximately 100-kDa glycoprotein bearing alpha-2,6-linked sialic acid in a colon cancer cell line (T84) in vitro. HST6N-1 mRNA levels were reduced approximately 80% by treatment (or = 24 h) with DOC or TPA but not by cholate, a primary bile acid. Treatment (24 h) with DOC or TPA decreased activity of this enzyme to 30% and 13% of control, respectively. These effects of DOC and TPA were transcriptional and were mediated by Ca2+ and protein kinase C, respectively. Thus DOC and TPA both downregulated, and did not upregulate, alpha-2,6-sialyltransferase expression in vitro, but by different transduction pathways. As colorectal tumors grow, their progressive removal from the fecal milieu that normally downregulates this enzyme may favor invasion and metastasis.

We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K + equilibrium potential (E K ) or Cl - equilibrium potential (E Cl ) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K + current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K + current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPγS), to pipettes stimulated large oscillating K + and inward currents. This occurred even when Ca 2+ was absent from the bath but not when the Ca 2+ chelator, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K + was replaced with Na + and cells were voltage clamped between E Na and E Cl , GTPγS activated oscillating Na + and Cl - currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] to pipettes activated large oscillating K + currents but only small inward currents. These results suggest that a carbachol-induced release of Ca 2+ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P 3 signaling pathway. In addition, this or another G protein activates Cl - current by directly gating Cl - channels to increase their sensitivity to Ca 2+ .

Salivary gland cell differentiation has been a recurring challenge for researchers as primary salivary cells show a loss of phenotype in culture. Particularly, parotid cells show a marked decrease in amylase expression, the loss of tight junction organization and proper cell function. Previously, Matrigel has been used successfully as an extracellular matrix; however, it is not practical for in vivo applications as it is tumorigenic. An alternative method could rely on the use of fibrin hydrogel (FH), which has been used extensively in biomedical engineering applications ranging from cardiovascular tissue engineering to wound-healing experiments. Although several groups have examined the effects of a three-dimensional (3D) environment on salivary cell cultures, little is known about the effects of FH on salivary cell cultures. The current study developed a 3D cell culture model to support parotid gland cell differentiation using a combination of FH and growth factor-reduced Matrigel (GFR-MG). Furthermore, FH polymerized with a combination of EGF and IGF-1 induced formation of 3D spheroids capable of amylase expression and an agonist-induced increase in the intracellular Ca(2+) concentration ([Ca(2+)]i) in salivary cells. These studies represent an initial step toward the construction of an artificial salivary gland to restore salivary gland dysfunction. This is necessary to reduce xerostomia in patients with compromised salivary function.

To cause colonization or infection, most bacteria grow in biofilms where differentiation and death of subpopulations is critical for optimal survival of the whole population. However, little is known about initiation of bacterial death under physiological conditions. Membrane depolarization has been suggested, but never shown to be involved, due to the difficulty of performing such studies in bacteria and the paucity of information that exists regarding ion transport mechanisms in prokaryotes. In this study, we performed the first extensive investigation of ion transport and membrane depolarization in a bacterial system. We found that HAMLET, a human milk protein-lipid complex, kills Streptococcus pneumoniae (the pneumococcus) in a manner that shares features with activation of physiological death from starvation. Addition of HAMLET to pneumococci dissipated membrane polarity, but depolarization per se was not enough to trigger death. Rather, both HAMLET- and starvation-induced death of pneumococci specifically required a sodium-dependent calcium influx, as shown using calcium and sodium transport inhibitors. This mechanism was verified under low sodium conditions, and in the presence of ionomycin or monensin, which enhanced pneumococcal sensitivity to HAMLET- and starvation-induced death. Pneumococcal death was also inhibited by kinase inhibitors, and indicated the involvement of Ser/Thr kinases in these processes. The importance of this activation mechanism was made evident, as dysregulation and manipulation of physiological death was detrimental to biofilm formation, a hallmark of bacterial colonization. Overall, our findings provide novel information on the role of ion transport during bacterial death, with the potential to uncover future antimicrobial targets.

Sjögren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sjögren's syndrome.

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.

Intracellular pH (pHc) was measured in the short-circuited epithelium of rabbit distal colon using H(+)-selective microelectrodes. pHc was 6.91 +/- 0.02 (SE) when the bath pH was 7.4. Intracellular HCO3- activity (acHCO3-) was estimated from these measurements to be 8 +/- 0 mM. When we replaced all Cl- in the tissue bathing solutions with the impermeant anion gluconate, pHc rose to 7.44 +/- 0.08 and acHCO3- increased to 30 +/- 6 mM. These results demonstrate that this tissue contains a Cl(-)-HCO3- exchange mechanism. During the Cl- replacement the apical membrane electrical potential difference hyperpolarized from -55 +/- 1 to -74 +/- 3 mV, suggesting that membrane ionic conductance had changed. Elevation of either the apical or basolateral membrane bathing solution K+ concentration produced a greater depolarization of membrane potential during Cl- replacement than when tissues were bathed in normal electrolyte solutions. In additional experiments, pHc was raised by lowering the bath CO2 concentration while the bath Cl- concentration was kept normal. Under these conditions, membrane potential hyperpolarized and was more sensitive to the elevation of bath K+ concentration than when pHc was normal. These results suggest that membrane K+ conductance in this tissue is increased by intracellular alkalinization.

KCNQ1 (Kv7.1 or KvLQT1) encodes the alpha-subunit of a voltage-gated potassium channel found in tissues including heart, brain, epithelia and smooth muscle. Tissue-specific characteristics of KCNQ1 current are diverse, due to modification by ancillary subunits. In heart, KCNQ1 associates with KCNE1 (MinK), producing a slowly activating voltage-dependent channel. In epithelia, KCNQ1 co-assembles with KCNE3 (Mirp2) producing a constitutively open channel. Chromanol 293B is a selective KCNQ1 blocker. We studied drug binding and frequency dependence of 293B on KCNQ1 and ancillary subunits expressed in Xenopus oocytes. Ancillary subunits altered 293B potency up to 100-fold (IC(50) for KCNQ1 = 65.4 +/- 1.7 microm; KCNQ1/KCNE1 = 15.1 +/- 3.3 microm; KCNQ1/KCNE3 = 0.54 +/- 0.18 microm). Block of KCNQ1 and KCNQ1/KCNE3 was time independent, but 293B altered KCNQ1/KCNE1 activation. We therefore studied frequency-dependent block of KCNQ1/KCNE1. Repetitive rapid stimulation increased KCNQ1/KCNE1 current biphasically, and 293B abolished the slow component. KCNQ1/KCNE3[V72T] activates slowly with a KCNQ1/KCNE1-like phenotype, but retains the high affinity binding of KCNQ1/KCNE3, demonstrating that subunit-mediated changes in gating can be dissociated from subunit-mediated changes in affinity. This study demonstrates the KCNQ1 pharmacology is significantly altered by ancillary subunits. The response of KCNQ1 to specific blockers will therefore be critically dependent on the electrical stimulation pattern of the target organ. Furthermore, the dissociation between gating and overall affinity suggests that mutations in ancillary subunits can potentially strongly alter drug sensitivity without obvious functional changes in gating behaviour, giving rise to unexpected side-effects such as a predisposition to acquired long QT syndrome.

We investigated prostanoid biogenesis by human colonic fibroblasts (CCD-18Co cells and nine primary fibroblast cultures) exposed to a primary (cholic, CA) or a secondary (deoxycholic, DCA) bile acid. Basal PGE2levels in CCD-18Co cultures and fibroblast strains initiated from normal and adenocarcinomatous colon, respectively, were 1.7 ± 0.3, 4.0 ± 2.0, and 15.0 ± 4.8 ng/mg protein. Peak levels 24 h after exposure to DCA (300 μM) rose, respectively, seven-, six- and sevenfold, but CA elicited no such responses. Increases in PGE2synthesis were preceded by sequential increases in PGH synthase-2 mRNA and protein expression and were fully prevented by a nonselective (indomethacin) or a selective (celecoxib) nonsteroidal anti-inflammatory drug. DCA, but not CA, caused abrupt, transient increases in fibroblast intracellular Ca2+concentration ([Ca2+]i) ∼1 min after exposure. Increased [Ca2+]iwas required for DCA-mediated induction of PGE2synthesis, and protein kinase C was a further essential component of this signaling pathway. Colonic fibroblasts may be a major target for prostanoid biogenesis induced by fecal bile acids and, potentially, other noxious actions of these agents.

Sex hormones have played a strong role in the history of KCNE potassium channel subunits. KCNE subunits were originally discovered as hormonally regulated potassium channel components isolated from uterine muscle (Pragnell et al. 1990). Despite this, the role that sex hormones and sex differences play in KCNE expression and function in non-gonadal tissues has remained relatively unexplored. Although initially misidentified as ‘MinK’, an independent pore forming subunit, KCNE1 and the KCNE family are now known to be ancillary subunits with significant influence on the stability and biophysical behaviour of potassium channel partners (Bett & Rasmusson, 2008). KCNE subunits are relatively small 57–150-amino-acid proteins that span the membrane with an extracellular N-terminal and an intracellular C-terminal. KCNE1 and KCNE3 are known to associate with the voltage-gated potassium channel KCNQ1 (KvLQT1, Kv7.1). The tissue-specific characteristics of electrophysiologically recorded KCNQ1 current are diverse, due to strong modification of KCNQ1 by these ancillary subunits. The KCNE1/KCNQ1 assembly produces a very slowly activating and deactivating current. In contrast, the KCNE3/KCNQ1 assembly produces a constituitively active current with very little voltage-dependent gating. It is commonly accepted in the field of ion channels that KCNQ1 and KCNE3 co-assemble in the epithelium of the colon to form a constitutively open potassium channel, underlying the basolateral membrane potassium current which controls fluid secretion (Schroeder et al. 2000). The initial studies of KCNQ1/KCNE3 were performed almost exclusively on male animals. In an elegant study on sexual dimorphism in rat colon in a recent issue of The Journal of PhysiologyAlzamora et al. (2011) confirm this finding in male rats, but surprisingly show that this is true only in male animals, not female animals. Alzamora et al. demonstrate that there are sex differences in the expression of the channel proteins between males and females, and furthermore that the relative expression differences change during the oestrous cycle. Alzamora et al. also demonstrate the short term hormonal regulation of the interaction between KCNQ1 and KCNE3, as oestrogen appears to uncouple KCNE3 from KCNQ1 but does not affect the interaction of KCNQ1 and KCNE1. This is a very significant finding since ancillary subunit expression can dramatically affect channel properties, and alter current magnitude, gating kinetics and pharmacological sensitivity. Indeed, Alzamora et al., demonstrate a 10 fold sex dependent change in the sensitivity to chromanol 293B: At the IC50 drug concentrations for chromanol 293B in males, the drug barely has an effect on the female colon. The hypothesized short-term mechanism of oestrogen action implies that there is a physiological site in the potassium current macromolecular complex which regulates dynamic subunit association by sex hormones. The oestrogen regulation of KCNE3 is removed by mutation of a serine on the intracellular side of KCNE3 to an alanine, S82A, which is the only phosphorylation site in KCNE, and therefore prevents phosphorylation. Alzamora et al. raise the exciting possibility that phosphorylation of S82 may mediate the effect of oestrogen on the interaction of KCNE3 and KCNQ1. This same phosphorylation site also modifies the behaviour of KCNE3/Kv3.4 channels (Abbott et al. 2006) opening up the possibility of a general KCNE3 mediated sex difference. The putative KCNE3 phosphorylation site is conserved across all five KCNEs, giving rise to potentially an even wider range of homologous binding sites with a similar mode of action. The impact of these findings is immense. KCNQ1 is found in an exceedingly diverse range of tissues including the heart, neuronal tissue, kidney, colonic crypt cells, pituitary, the stria vascularis and the vestibular dark cells of the cochlea, stomach, small intestine, liver, thymus, exocrine pancreas, prostate, skeletal muscle, airway epithelia, ovarian tissue, testis, uterus, and placenta. While considerable attention has been given to understanding the tissue specific effects of the KCNE subunits on KCNQ1, the results of Alzamora et al. clearly show that studying the effects of sex differences on KCNQ1 complexes in these varied tissues will yield many interesting findings. Further, these results beautifully demonstrate the more general need to pay careful attention to the sex of the animals under study, and to determine whether physiological responses are sex dependent. In the last several issues of The Journal of Physiology where animal tissue was used, 13 papers examined only the physiology of male animals and one paper focused on the physiology of females alone. Seventeen other papers used animals of unspecified sex, and five papers reported results from both sexes. The careful analysis of the sex-dependent nature of the physiological responses observed by Alzamora et al. suggest that caution should be exercised when extrapolating a study performed on one sex only, to both sexes. This study by Alzamora et al. adds to a growing body of work that indicates there can be striking sex differences in gene expression, cellular regulation and pharmacological sensitivity in non-gonadal organs. The observed long-term and short-term regulation by sex hormones raises exciting new possibilities. Sex differences are complex, but fundamental to our nature. This study underscores the potential wealth of information and novel signalling pathways that may be uncovered by studying sex differences.

PURPOSE Carbonic anhydrases play a central buffering role in current models of fluid transport in corneal endothelium, but in humans, clinical use of carbonic anhydrase inhibitors (CAIs) for the management of glaucoma does not cause corneal swelling. This study compares species differences in response to CAIs in human versus bovine corneal endothelial transport. METHODS Short-circuit current (Isc) measurements were performed on bovine and human corneal endothelium under identical conditions. The effects of four CAIs (acetazolamide, brinzolamide, dorzolamide, and ethoxzolamide) were measured. Endothelial expression of carbonic anhydrase II and IV was evaluated by immunofluorescence microscopy. Functional presence of carbonic anhydrase activity was determined using the Hansson's cobalt sulfide histochemical method. RESULTS All four CAIs decreased bovine Isc (% change in Isc: acetazolamide, -21.0 ± 9.5, n = 8; brinzolamide, -35.5 ± 13.5, n = 9; dorzolamide, -33.6 ± 7.2, n = 8; ethoxzolamide, -35.3 ± 12.9, n = 8). That decrease was not present in humans (% change in Isc: acetazolamide, 16.2 ± 20.1, n = 3; brinzolamide, 6.7 ± 13.9, n = 3; dorzolamide, 8.0 ± 20.4, n = 3; ethoxzolamide, -4.8 ± 10.3, n = 2). Despite no functional effect of CAIs on Isc, both carbonic anhydrase II and IV were present in human corneal endothelium by immunofluorescence microscopy. Histochemical analysis of human corneal endothelium revealed functionally active carbonic anhydrase activity inhibited by brinzolamide. CONCLUSIONS Carbonic anhydrase facilitates ion transport impacting the corneal endothelial Isc in bovine but not human corneal endothelium, despite its presence and functional activity in human tissue. This finding supports the clinical observation of no corneal swelling in humans administered CAIs and suggests that alternative ion transport mechanisms may be operational in corneal endothelium of different species.

The stimulation of epithelial chloride secretion by hormones and neurotransmitters involves the activation of apical membrane chloride channels. The regulation of chloride current by acetylcholine in the T-84 colonic cell line was investigated using single-channel patch-clamp techniques. Treatment with carbachol resulted in the stimulation of transient chloride currents in 18 of 32 previously quiescent patches. Lack of resolvable single-channel openings suggests that single-channel conductance is less than 5-pS. Of 18 responsive patches, 4 showed multiple current oscillations. Treatment of the cells with AlF4 – activated sustained chloride currents, suggesting that G proteins are involved. In excised patches, chloride current was markedly sensitive to free Ca2+ concentrations between 500 and 1000 nM. Time-dependent activation and inactivation of chloride current occurred at +60 and –60 mV. These results indicate that the chloride channels responsible for cholinergic activation of chloride conductance in the T-84 colonic cell line are members of the very low conductance family of chloride channels.

109 Background: Survivorship education may facilitate coordination of care between the patient and health care providers, increase adherence to health maintenance recommendations, and improve outcomes. Approximately 80% of Americans regularly use the internet. Interactive seminars (webinars) may offer an efficient tool to reach a diverse audience of cancer survivors, caregivers, and providers. Methods: From September 2012-April 2013, we offered 3 free 1-hour webinars advertised via social media, print flyers, email, newsletters, and advocacy and support groups. Topics included next steps after breast cancer treatment, integrative medicine, and nutrition. Participants registered online or by phone. Format consisted of a 30-minute lecture followed by live Q&A with electronically submitted questions. Demographic data were collected at registration and participants were asked to complete a brief 9-question survey after the webinar. Results: 728 participants registered for at least 1 webinar (median age 56, range 18-81 years; 14% African American). 681 participants were U.S.-based and 47 joined from 21 other countries. Participants self-identified as a cancer survivor (71%), provider (20%), caregiver (8%), or other (2%). Most (79%) had at least a high school education. Among the 184 (25%) who completed the post-webinar survey, 86% reported that information was relevant to situation, 85% said they had the opportunity to ask questions, 94% felt speakers were knowledgeable, and 90% were satisfied with the information quality. Participants ranked one-on-one care followed by webinars as their preferred way to receive survivorship information, above other tools like videos, websites, and handouts. Conclusions: As part of a multidisciplinary approach to breast cancer survivorship care, webinars are a feasible modality to deliver quality health information. Limitations include overall generalizability with a potential selection bias of those who may be more educated than the general population, have access to the internet, and feel comfortable with this technology. Nonetheless, data suggest that this format may allow us to reach a diverse audience that appears receptive and satisfied with survivorship education provided in an electronic format.

Timothy Syndrome (TS) arises from a point mutation in the human voltage-gated L-type Ca2+ channel (Cav1.2). TS is associated with cardiac arrhythmias and sudden cardiac death, as well as congenital heart disease, impaired cognitive function, and autism spectrum disorders. TS results from a de novo gain-of-function mutation which affects the voltage dependent component of Cav1.2 inactivation. We created a knock-in TS mouse. No homozygous TS mice survived, but heterozygous TS2-NEO mice (with the mutation and the neocassette in situ) had a normal outward appearance and survived to reproductive age. Previously, we have demonstrated that these mice exhibit the triad of Autistic traits. In this paper we document other aspects of these mice including Cav1.2 isoform expression levels, normal physical strength, brain anatomy and a marked propensity towards self-injurious scratching. Gross brain anatomy was not markedly different in TS2-NEO mice compared to control littermates, and no missing structures were noted. The lack of obvious changes in brain structure is consistent with theTS2-NEO mice may provide a significant tool in understanding the role of calcium channel inactivation in both cardiac function and brain development.