A nitrocellulose membrane based solid phase enzyme immunoassay for detecting polioviruses in stool

An enzyme immunoassay was developed with nitrocellulose membrane as the solid phase support built in a 96 well porous polystyrene plate. Monoethanolamine was found to be a satisfactory and better blocking agent than skimmed milk. Up to 10(4.28) TCID50 poliovirus particles/0.1 ml of stool collected in 10% skimmed milk could be detected depending on the initial titer of the antigen specific capturing serum/IgG immobilized on nitrocellulose membrane. Percentage of skimmed milk in the transport medium, composition and pH of the dilution buffer and chloroform treatment of the stool specimens before the test were important determinants of the specificity of the test. Polyvinyl affinity membrane did not appear to be superior to nitrocellulose membrane as a solid phase support.     

A functional clotting assay to monitor the hirudin dosage.

Hirudin, a direct thrombin inhibitor, has potential advantages over indirect thrombin inhibitors and is increasingly used in clinical settings. There are, however, large variations in individual responses to this drug and no recognized clinical laboratory tests used to monitor its anticoagulant effects. We evaluated the use of the thromboelastograph, a common clinical coagulation instrument, to monitor the effects of hirudin in vitro. We developed a novel, whole blood clotting assay that utilizes the tissue factor stimulating properties of mercuric ion to measure the anticoagulant potential of therapeutic doses of hirudin. At doses equivalent to those found in the therapeutic range, the thromboelastograph was capable of showing significant changes when compared with control and different concentrations of hirudin (P < 0.05). A linear relationship was observed between increasing concentrations of recombinant hirudin and clotting times. In conclusion, the use of this test system warrants further investigation for monitoring hirudin.     

Marine phosphatidylcholine suppresses 1,2-dimethylhydrazine-induced colon carcinogenesis in rats by inducing apoptosis

In vitro and animal studies indicate that n-3 polyunsaturated fatty acids (PUFAs) suppress carcinogenesis. This study presents a new insight on effectiveness of marine phospholipids for suppression of colon carcinogenesis. The purpose of this study was to investigate growth inhibition and apoptosis inducing effects of n-3 PUFA in the form of marine phosphatidylcholine (PC) on chemically induced (1,2-dimethylhydrazine) colon cancer in rats. Growth inhibition of Caco-2 cells was determined by colorimetric sodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) dye reduction assay. For animal studies, the rats were fed 5 different diets containing docosahexaenoic acid (DHA)-ethyl ester, eicosapentaenoic acid (EPA)-ethyl ester, squid meal PC (rich in DHA), starfish PC (rich in EPA), and corn oil. The 1,2-dimethylhydrazine (30 mg/kg) or saline was injected 48 hours before the experiment. Rats were anesthetized, and apoptotic as well as mitotic cells in crypt were counted based on morphological criteria in isolated crypts. Squid meal and starfish PC potently inhibited the growth of Caco-2 cells. The experimental diets containing n-3 PUFA suppressed colon cancer in rats. Rats that consumed diets containing DHA-ethyl ester, EPA-ethyl ester, squid meal PC, and starfish PC showed increased apoptosis (P < .01) and suppressed proliferation. These results suggest that marine PC-containing diets might be an effective dietary protective factor against colon cancer.     

Induced Abortions and Unintended Pregnancies in Pakistan

During the past decade, unmet need for family planning has remained high in Pakistan and gains in contraceptive prevalence have been small. Drawing upon data from a 2012 national study on postabortion‐care complications and a methodology developed by the Guttmacher Institute for estimating abortion incidence, we estimate that there were 2.2 million abortions in Pakistan in 2012, an annual abortion rate of 50 per 1,000 women. A previous study estimated an abortion rate of 27 per 1,000 women in 2002. After taking into consideration the earlier study's underestimation of abortion incidence, we conclude that the abortion rate has likely increased substantially between 2002 and 2012. Varying contraceptive‐use patterns and abortion rates are found among the provinces, with higher abortion rates in Baluchistan and Sindh than in Khyber Pakhtunkhwa and Punjab. This suggests that strategies for coping with the other wise uniformly high unintended pregnancy rates will differ among provinces. The need for an accelerated and fortified family planning program is greater than ever, as is the need to implement strategies to improve the quality and coverage of postabortion services.     

Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy

Endothelin 1 (ET-1), a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. We tested whether ET-1 generated by cardiac myocytes in vivo is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic ET-1-null mice, we used the Cre/loxP system to generate mice with cardiac myocyte-specific disruption of the ET-1 gene. We used the alpha-myosin heavy chain promoter to drive expression of Cre and were able to obtain 75% reduction in ET-1 mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation, in vivo, for 24 h with tri-iodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted ET-1 allele compared to siblings with an intact ET-1 gene. Moreover, in vivo MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 27% increase in mice with an intact ET-1 gene. A reduced hypertrophic response was confirmed by planimetry of cardiac myocytes. We conclude that ET-1, produced locally by cardiac myocytes, and acting in a paracrine/autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone.     

Determinants of right ventricular failure in patients admitted with acute left heart failure

Pulmonary hypertension, which may lead to right ventricular (RV) failure, increases with left ventricular (LV) diastolic dysfunction severity. The prevalence and determinants of RV failure were analyzed in 120 patients admitted with acute left heart (LH) failure. Patients were divided into RV failure (n=50) and non-RV failure (n=70) groups. The prevalence of RV failure was found to be 42%. In both groups, two thirds of the patients had isolated LV diastolic dysfunction and the rest had combined LV systolic and diastolic dysfunction. Patients in the RV failure group were characterized by higher LV diastolic grade (2.2 ± 0.6 vs 1.84 ± 0.7; P=.0070), pulmonary artery systolic pressure (PASP; 57.8 ± 15.3 vs 50.14 ± 12.1 mm Hg; P=.0028), right atrial enlargement (92% vs 25.7%; P=.000001), and more-than-moderate tricuspid regurgitation (58% vs 27.1%; P=.0006). RV failure is a frequent finding in patients with advanced LH failure. It is strongly associated with the severity of LV diastolic dysfunction and the severity of PASP.     

Protein Kinase CK1αLS Promotes Vascular Cell Proliferation and Intimal Hyperplasia

Protein kinase CK1α regulates several fundamental cellular processes including proliferation and differentiation. Up to four forms of this kinase are expressed in vertebrates resulting from alternative splicing of exons; these exons encode either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein. Whereas the L-insert is known to target the kinase to the nucleus, the functional significance of nuclear CK1αLS has been unclear. Here we demonstrate that selective L-insert-targeted short hairpin small interfering RNA-mediated knockdown of CK1αLS in human vascular endothelial cells and vascular smooth muscle cells impairs proliferation and abolishes hydrogen peroxide-stimulated proliferation of vascular smooth muscle cells, with the cells accumulating in G₀/G₁. In addition, selective knockdown of CK1αLS in cultured human arteries inhibits vascular activation, preventing smooth muscle cell proliferation, intimal hyperplasia, and proteoglycan deposition. Knockdown of CK1αLS results in the harmonious down-regulation of its target substrate heterogeneous nuclear ribonucleoprotein C and results in the altered expression or alternative splicing of key genes involved in cellular activation including CXCR4, MMP3, CSF2, and SMURF1. Our results indicate that the nuclear form of CK1α in humans, CK1αLS, plays a critical role in vascular cell proliferation, cellular activation, and hydrogen peroxide-mediated mitogenic signal transduction.A key morphological distinction between vertebrates and invertebrates is the presence of a closed endothelial-lined vascular system in the vertebrates.¹ Activation of the cells comprising the vertebrate vasculature results in cellular proliferation, enhanced proteoglycan deposition, and secretion of growth factors and cytokines.^2,3,4 Such vascular activation is an important process in both vascular development and in vascular diseases such as atherosclerosis and postangioplasty restenosis. Thus, an understanding of the vertebrate-specific signaling pathways regulating vascular cell activation is of high importance.Protein kinase CK1α regulates several fundamental cellular processes including proliferation and differentiation.⁵ Up to four different forms of the kinase exist owing to the alternative splicing of exons encoding either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein.^6,7,8,9 Protein kinase CK1α itself is highly conserved among all metazoans. However, the exon encoding the nuclear localizing L-insert is restricted to vertebrates.¹⁰ Whereas vertebrates may contain up to four different splice forms of CK1α, humans are thought to only express three forms: CK1α, CK1αS, and CK1αLS, which are also referred to as CK1α1, CK1α2, and CK1α3, respectively. In the nucleus, CK1αLS probably plays a role in pre-mRNA processing and alternative splicing based on its ability to phosphorylate the highly abundant vertebrate-specific pre-mRNA binding protein heterogeneous nuclear ribonucleoprotein C (hnRNP-C)^10,11,12 and its localization to nuclear speckles,⁶ sites of accumulation of pre-mRNA processing factors.Within the vessel wall, hydrogen peroxide (H₂O₂) plays important roles in mediating vascular activation resulting from diverse stimuli including altered flow, growth factors, cytokines, and vascular injury.^13,14 In fact, vertebrate cells are known to proliferate in response to low concentrations of H₂O₂.¹⁵ Low levels of H₂O₂ are generated by vertebrate cells in response to growth factor-mediated signaling, and this mitogenic H₂O₂ activates CK1αLS, which then phosphorylates hnRNP-C.^10,11 It is known that hnRNP-C modulates the expression of several genes regulating cell growth and survival, including platelet-derived growth factor B chain (PDGF-B),¹⁶ c-myc,¹⁷ p53,¹⁸ the X-chromosome-linked inhibitor of apoptosis,¹⁹ and the urokinase plasminogen activator receptor.²⁰ Thus, CK1αLS phosphorylation of hnRNP-C may promote H₂O₂-stimulated vertebrate cell growth. However, in the cytoplasm, cytosolic forms of CK1α (CK1α and CK1αS) play important roles inhibiting key proliferative signaling pathways involving both wnt/β-catenin and the nuclear factor of activated T-cells.^21,22 Thus, it has been unclear whether H₂O₂-activated CK1αLS in the nucleus is promoting H₂O₂-stimulated growth or is, in fact, a compensatory counter-regulatory pathway. Here we demonstrate by selective and stable knockdown of CK1αLS that the kinase is, in fact, an important positive regulator of vascular activation and H₂O₂ mitogenic signaling.