Layered Chiral Active Matter: Beyond Odd Elasticity.

In equilibrium liquid crystals, chirality leads to a variety of spectacular three-dimensional structures, but chiral and achiral phases with the same broken continuous symmetries have identical long-time, large-scale dynamics. In this Letter, starting from active model H^{*}, the general hydrodynamics of a pseudoscalar in a momentum-conserving fluid, we demonstrate that chirality qualitatively modifies the dynamics of layered liquid crystals in active systems in both two and three dimensions due to an active "odder" elasticity. In three dimensions, we demonstrate that the hydrodynamics of active cholesterics differs fundamentally from smectic-A liquid crystals, unlike their equilibrium counterpart. This distinction can be used to engineer a columnar array of vortices, with an antiferromagnetic vorticity alignment, that can be switched on and off by external strain. A two-dimensional chiral layered state-an array of lines on an incompressible, freestanding film of chiral active fluid with a preferred normal direction-is generically unstable. However, this instability can be tuned in easily realizable experimental settings when the film is either on a substrate or in an ambient fluid.

Effect of a Family Media Use Plan on Media Rule Engagement Among Adolescents: A Randomized Clinical Trial.

Importance: The American Academy of Pediatrics recommends that all families use a family media use plan to select and engage with media rules. To date, the effectiveness of this tool in promoting adolescent media rule engagement is unknown. Objective: To test the effect of a family media use plan on media rule engagement in adolescents. Design, Setting, and Participants: This randomized clinical trial with parallel design used the online Qualtrics platform for recruitment, data collection, and intervention delivery. Parents and their children (aged 12 to 17 years) who spoke and read in the English language were recruited, enrolled, and randomized to either the intervention or control group. Parent-adolescent dyads in both groups completed baseline surveys individually, and the dyads in the intervention group completed the family media use plan survey. Baseline recruitment was conducted from April 8, 2019, to May 1, 2019, and follow-up surveys were completed between June 11, 2019, and July 2, 2019. Interventions: The American Academy of Pediatrics family media use plan. Main Outcomes and Measures: The primary outcome was media rule engagement reported by adolescents. Media rules were extracted from the family media use plan, and adolescents rated each rule (using Likert scales) according to whether the rule was present or followed in their home. Secondary outcomes were adolescent-perceived technology importance and changes in sleep, physical activity, and depression. Results: A total of 1520 parent-adolescent dyads were enrolled in the trial and randomized to either the intervention or control group. Adolescents had a mean (SD) age of 14.5 (1.6) years, and 789 were female (51.9%) and 1027 were White (67.6%) individuals. Parents had a mean (SD) age of 44.1 (8.5) years, and 995 were women (65.5%) and 1021 were White individuals (67.2%). For media rule engagement, the between-group difference was -0.1 (95% CI, -1.1 to 0.9). Conclusions and Relevance: This randomized clinical trial found that completing a family media use plan did not lead to statistically significant changes in media rule engagement for adolescents in the intervention group. Future studies should consider revising the family media use plan and exploring the importance of technology as an intervention outcome. Trial Registration: ClinicalTrials.gov Identifier: NCT03881397.

Arsenic-tolerant, arsenite-oxidising bacterial strains in the contaminated soils of West Bengal, India.

As biological agents represent an affordable alternative to costly metal decontamination technologies, we isolated arsenic (As) oxidising bacteria from the As-contaminated soils of West Bengal, India. These strains were closely related to various species of Bacillus and Geobacillus based on their 16S rRNA gene sequences. They were found to be hyper-resistant to both As(V) (167-400 mM) and As(III) (16-47 mM). Elevated rates of As(III) oxidation (278-1250 µM h(-1)) and arsenite oxidase activity (2.1-12.5 nM min(-1) mg(-1) protein) were observed in these isolates. Screening identified four strains as superior As-oxidisers. Among them, AMO-10 completely (100%) oxidised 30 mM of As(III) within 24 h. The presence of the aoxB gene was confirmed in the screened isolates. Phylogenetic tree construction based on the aoxB sequence revealed that two strains, AGO-S5 and AGH-02, clustered with Achromobacter and Variovorax, whereas the other two (AMO-10 and ADP-25) remained unclustered. The increased rate of As(III) oxidation by these native strains might be exploited for the remediation of As in contaminated environments. Notably, this study presents the first correlation regarding the presence of the aoxB gene and As(III) oxidation ability in Geobacillus stearothermophilus.

Efficacy of indigenous soil microbes in arsenic mitigation from contaminated alluvial soil of India.

Selected arsenic-volatilizing indigenous soil bacteria were isolated and their ability to form volatile arsenicals from toxic inorganic arsenic was assessed. Approximately 37 % of AsIII (under aerobic conditions) and 30 % AsV (under anaerobic conditions) were volatilized by new bacterial isolates in 3 days. In contrast to genetically modified organism, indigenous soil bacteria was capable of removing 16 % of arsenic from contaminated soil during 60 days incubation period while applied with a low-cost organic nutrient supplement (farm yard manure).

Arsenic accumulating bacteria isolated from soil for possible application in bioremediation.

Twenty six arsenic (As) resistant bacterial strains were isolated from As contaminated paddy soil of West Bengal, India. Among them, 10 isolates exhibited higher As resistance capacity and could grow in concentration of 12000 mg I-1 of arsenate (AsV) and 2000 mg l-1 of arsenite (Aslll) in growing medium. Maximum growth was observed at 1000 mg I-1and 100 mg I-1 in case of AsV and Asll respectively. Results of incubation study carried out in basal salt minimal media (BSMY) containing 25 mg l-1 of AsV and Aslll separately showed that the isolates could accumulate 1.03 - 6.41 mg I-1 of AsV and 2.0 - 7.6 mg l-1 of Aslll from the media. The bacterial isolate AGH-21 showed highest As accumulating capacity both for AsV (25.64%) and Aslll (30.4%) under laboratory conditions. The isolates AGH-21 (NCBI accession no: HQ834295) showed highest sequence similarity (98%) with Bacillus sp. and could be used as a potential bioremediator in future to combat with arsenic toxicity.

Ligand and protein fishing with heat shock protein 90 coated magnetic beads.

Heat shock protein 90alpha (Hsp90alpha) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90alpha has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90alpha ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90alpha coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.

In vitro effects of estradiol, dydrogesterone, tamoxifen and cyclophosphamide on proliferation vs. death in human breast cancer cells.

The effects of 17 beta-estradiol, dihydrodydrogesterone, tamoxifen and cyclophosphamide upon parameters of cell maturation (Mucine1 expression), cell proliferation (Cyclin D1 expression) and apoptosis (loss of nuclear DNA) were studied in estrogen receptor positive (ER+) and negative (ER-) human breast cancer cells. Tamoxifen was the most potent inducer of apoptosis in ER+ and ER- breast cancer cells. 17 beta-estradiol in a concentration of 10(-6) M induced proliferation in ER+ cells after 144 h. incubation, while equimolar co-incubation with dihydrodydrogesterone prevented this effect and even induced a significant increase of cell death. It is speculated that the continuous use of combined 17 beta-estradiol plus dihydrodydrogesterone might be given as hormone replacement therapy without increased risk of breast cancer and even may reduce the relapse rate in breast cancer patients.

Engaging ethnically diverse teens in a substance use prevention advocacy program.

Teen Activists for Community Change and Leadership Education is designed to engage high school students living in low-income neighborhoods in community advocacy efforts to transform their schools and communities so they do not reinforce use of alcohol, tobacco, and other drugs. This nine month intervention for 116 freshmen and sophomores in and near San Jose, California consisted of 30-90 minute meetings. Social cognitive constructs of sense of community, perceived self-efficacy, outcome expectancies, incentive value, policy control, and leadership competence guided the program. No changes in individual use of alcohol, tobacco, and other drugs were observed by the end of the program, but improvements in community involvement and self-perception of many of the constructs were observed.

Akt-dependent antiapoptotic action of insulin is sensitive to farnesyltransferase inhibitor.

CHO cells expressing the human insulin receptors (IR) were used to evaluate the effect of the potent farnesyltransferase inhibitor, manumycin, on insulin antiapoptotic function. Cell treatment with manumycin blocked insulin's ability to suppress pro-apoptotic caspase-3 activity which led to time-dependent proteolytic cleavage of two nuclear target proteins. The Raf-1/MEK/ERK cascade and the serine/threonine protein kinase Akt are two survival pathways that may be activated in response to insulin. We tested the hypothesis that inhibition of farnesylated Ras was causally related to manumycin-induced apoptosis and showed that the response to manumycin was found to be independent of K-Ras function because membrane association and activation of endogenous K-Ras proteins in terms of GTP loading and ERK activation were unabated following treatment with manumycin. Moreover, blocking p21Ras/Raf-1/MEK/ERK cascade by the expression of a transdominant inhibitory mSOS1 mutant in CHO-IR cells kept cells sensitive to the antiapoptotic action of insulin. Insulin-dependent activation of Akt was blocked by 4 h treatment with manumycin (P < 0.01), a kinetic too rapid to be explained by Ras inhibition. This study suggests that the depletion of short-lived farnesylated proteins by manumycin suppresses the antiapoptotic action of insulin at least in part by disrupting Akt activation but not that of the K-Ras/Raf-1/ERK-dependent cascade.

Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein.

The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains cysteine (Cys) residues whose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctional cross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins that possess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complex between the wild-type human receptor beta-subunit and molecule X, a thiol-reactive membrane-associated protein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulin had only a modest effect in this process. Western blot analysis revealed that the receptor alpha-subunit was not present in the beta-X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, that lacks the equivalent of human Cys(981), failed to react with BMH. Finally, no covalent association between IR beta-subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treated cells expressing the wild-type human IR. These results demonstrate a striking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulated conditions.

Discrete region of the insulin receptor carboxyl terminus plays key role in insulin action.

In the present study, we attempted to determine the importance of a 23-amino-acid sequence within the carboxyl terminus of the human insulin receptor (IR) molecule in modulating insulin action in Chinese hamster ovary cells. Stable expression of a minigene encoding the receptor fragment led to an increase in insulin-induced IR autophosphorylation that was 2.4-fold higher when compared to that of IR-expressing cells transfected with empty vector. Insulin-stimulated downstream signaling was also significantly elevated in cells expressing the minigene. It was found that expression of the minigene had no effect toward insulin-like growth factor I receptor kinase activity and function. These results indicate that the IR carboxyl terminus contains a motif that acts as a physiologic modulator of insulin signaling. J. Cell. Biochem. 78:160-169, 2000. Published 2000 Wiley-Liss, Inc.

Refined genetic and comparative physical mapping of the canine copper toxicosis locus.

Recently, the copper toxicosis (CT) locus in Bedlington terriers was assigned to canine chromosome region CFA10q26, which is homologous to human chromosome region HSA2p13-21. A comparative map between CFA10q21-26 and HSA2p13-21 was constructed by using genes already localized to HSA2p13-21. A high-resolution radiation map of CFA10q21-26 was constructed to facilitate positional cloning of the CT gene. For this map, seven Type I and eleven Type II markers were mapped. Using homozygosity mapping, the CT locus could be confined to a 42.3 cR(3000) region, between the FH2523 and C10.602 markers. On the basis of a partial BAC contig, it was estimated that 1-cR(3000) is equivalent to approximately 210 kb, implying that the CT candidate region is therefore estimated to be about 9 Mb.

Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells.

In this study, we used maleimidobutyrylbiocytin to examine possible alteration that may occur in the redox state of the insulin receptor (IR) sulfhydryl groups in response to reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC). Short-term treatment of intact cells expressing large numbers of IR with GSH or NAC led to a rapid and reversible reduction of IR alpha-subunit disulfides, without affecting the receptor beta-subunit thiol reactivity. The overall integrity of the oligomeric structure of IR was maintained, indicating that neither class I nor class II disulfides were targeted by these agents. Similar findings were obtained in cells transfected with IR mutants lacking cysteine524, one of the class I disulfides that link the two IR alpha-subunits. Membrane-associated thiols did not participate in GSH- or NAC-mediated reduction of IR alpha-subunit disulfides. No difference in insulin binding was observed in GSH-treated cells; however, ligand-mediated increases in IR autophosphorylation, tyrosine phosphorylation of cellular substrates, and dual phosphorylation of the downstream target mitogen-activated protein kinase were inhibited at concentrations of GSH (10 mM or greater) that yielded a significant increase in IR alpha-subunit thiol reactivity. GSH did not affect IR signaling in the absence of insulin. Our results provide the first evidence that the IR alpha-subunit contains a select group of disulfides whose redox status can be rapidly altered by the reducing agents GSH and NAC.

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

Antiapoptotic signaling by the insulin receptor in Chinese hamster ovary cells.

We have sought to determine whether insulin can promote cell survival and protect Chinese hamster ovary (CHO) cells from apoptosis induced by serum starvation. Low concentrations of insulin were antiapoptotic for cells overexpressing wild-type insulin receptors but not in cells transfected with kinase-defective insulin receptor mutants that lacked a functional ATP binding site. However, treatment with orthovanadate (50 microM), a widely used tyrosine phosphatase inhibitor, led a dramatic reduction in internucleosomal DNA fragmentation in both cell lines. Cells transfected with truncated receptor mutants in either the juxtamembrane or C-terminal domain were as responsive as cells overexpressing wild-type receptors in mediating insulin antiapoptotic protection. The mechanisms underlying insulin antiapoptotic protection were investigated using a variety of pharmacological tools known to inhibit distinct signaling pathways. The phosphatidylinositol-3' kinase inhibitors wortmannin and LY294002 had only a modest influence whereas blocking protein farnesylation with manumycin severely disrupted the antiapoptotic capacity of the insulin receptor. Of interest, cells gained antiapoptotic potential following inhibition of extracellular signal-regulated kinase activation with the pharmacological agent PD98059. Insulin induced MKK3/MKK6 phosphorylation and activation of p38 MAP kinase whose activity was inhibited with SB203580. However, the inhibition of p38 MAP kinase had no effect on the protection offered by insulin. We conclude that the antiapoptotic function of the insulin receptor requires intact receptor kinase activity and implicates a farnesylation-dependent pathway. Increase in cellular phosphotyrosine content, however, triggers antiapoptotic signal that may converge downstream of the insulin receptor.

Clamp for coronary artery operations.

Aortocoronary bypass grafting is an accepted procedure for ischemic heart disease. Proper visualization of the coronary artery is mandatory for good surgical anastomosis. This is essential when a coronary operation is performed without cardioplegia or in surgical procedures without bypass support. For better visualization of a coronary artery, we are presenting a coronary artery clamp. We have used this clamp in minimally invasive coronary artery operations to achieve a bloodless field.

Inhibition of the transmembrane protein tyrosine phosphatase lar by 3S-peptide-I enhances insulin receptor phosphorylation in intact cells.

3S-peptide-I, a tris-sulfotyrosyl dodecapeptide that corresponds to the major autophosphorylation domain within the insulin receptor beta-subunit, selectively enhances insulin signal transduction by specifically inhibiting dephosphorylation of the insulin receptor catalyzed by protein tyrosine phosphatases (PTPases). Because of the potential role of the transmembrane PTPase LAR in the regulation of insulin signaling, we assessed the effect of 3S-peptide-I on recombinant LAR PTPase activity and in McA-RH7777 rat hepatoma cells overexpressing full-length LAR protein (McA4B/LAR). 3S-peptide-I significantly reduced insulin receptor dephosphorylation by recombinant LAR (p < 0.001) while blocking dephosphorylation of the insulin receptor by approximately 72% in semi-permeabilized McA4B/LAR cells (p < 0.001). Increased LAR expression resulted in 40% reduction in ligand-mediated phosphorylation of the insulin receptor compared with null vector control (p < 0.001). However, treatment of intact McA4B/LAR cells with a fatty acid derivative of 3S-peptide-I (50 microM) led to an enhancement of insulin-stimulated receptor phosphorylation by 89% (p < 0.001). As a result, control and McA4B/LAR cells showed comparable steady-state levels of insulin receptor phosphorylation in the presence of insulin. These findings provide evidence that 3S-peptide-I may improve insulin responsiveness in intact cells by inhibiting LAR, an enzyme whose activity has been implicated in the pathogenesis of insulin resistance.

Specific inhibition of insulin receptor dephosphorylation by a synthetic dodecapeptide containing sulfotyrosyl residues as phosphotyrosyl mimetic.

We have synthesized a tris-sulfotyrosyl dodecapeptide (3S-peptide-I) that corresponds to the major autophosphorylation domain within the insulin receptor beta-subunit and showed that it potently inhibited insulin receptor dephosphorylation by protein tyrosine phosphatases (PTPases) in vitro. 3S-peptide-I also inhibited tyrosine dephosphorylation of a synthetic peptide by the recombinant PTPase PTP-1B, indicating that 3S-peptide-I interacts directly with PTPase, causing its inactivation. The peptide had no effect on the activity of serine/threonine phosphatases, PP-1 and PP-2A, or alkaline phosphatase. Furthermore, we found that the introduction of a N-stearyl derivative of 3S-peptide-I in CHO/HIRc cells caused a significant increase in insulin-stimulated phosphorylation of the insulin receptor. In contrast, ligand-stimulated phosphorylation of epidermal growth factor (EGF) receptor in CHO cells overexpressing EGF receptors was not affected by the presence of N-stearyl-3S-peptide-I. These data suggest that by inhibiting dephosphorylation of the insulin receptor in intact cells, 3S-peptide-I may specifically enhance insulin signalling.