SUMMARY The sandwich estimator in generalized estimating equations (GEE) approach underestimates the true variance in small samples and consequently results in inflated type I error rates in hypothesis testing. This fact limits the application of the GEE in cluster-randomized trials (CRTs) with few clusters. Under various CRT scenarios with correlated binary outcomes, we evaluate the small sample properties of the GEE Wald tests using bias-corrected sandwich estimators. Our results suggest that the GEE Wald z test should be avoided in the analyses of CRTs with few clusters even when bias-corrected sandwich estimators are used. With t-distribution approximation, the Kauermann and Carroll (KC)-correction can keep the test size to nominal levels even when the number of clusters is as low as 10, and is robust to the moderate variation of the cluster sizes. However, in cases with large variations in cluster sizes, the Fay and Graubard (FG)-correction should be used instead. Furthermore, we derive a formula to calculate the power and minimum total number of clusters one needs using the t test and KC-correction for the CRTs with binary outcomes. The power levels as predicted by the proposed formula agree well with the empirical powers from the simulations. The proposed methods are illustrated using real CRT data. We conclude that with appropriate control of type I error rates under small sample sizes, we recommend the use of GEE approach in CRTs with binary outcomes due to fewer assumptions and robustness to the misspecification of the covariance structure.

(E.M.); 0000-0001-7707-7776 (J.-P.R.); 0000-0002-5725-885X (J.P.).Our knowledge of the factors mediating ethylene-dependent ripening of climacteric fruit remains limited. The transcription of ethylene-regulated genes is mediated by ethylene response factors (ERFs), but mutants providing information on the specific role of the ERFs in fruit ripening are still lacking, likely due to functional redundancy among this large multigene family of transcription factors. We present here a comprehensive expression profiling of tomato (Solanum lycopersicum) ERFs in wild-type and tomato ripening-impaired tomato mutants (Never-ripe [Nr], ripening-inhibitor [rin], and non-ripening [nor]), indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening while 28 display a ripeningassociated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Among the 19 ERFs exhibiting the most consistent up-regulation during ripening, the expression of 11 ERFs is strongly down-regulated in rin, nor, and Nr tomato ripening mutants, while only three are consistently up-regulated. Members of subclass E, SlERF.E1, SlERF.E2, and SlERF.E4, show dramatic down-regulation in the ripening mutants, suggesting that their expression might be instrumental in fruit ripening. This study illustrates the high complexity of the regulatory network connecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene-and RIN/NOR-dependent ripening.The plant hormone ethylene is involved in a wide range of developmental processes and physiological responses such as flowering, fruit ripening, organ senescence, abscission, root nodulation, seed germination, programmed cell death, cell expansion, and responses to abiotic stresses and pathogen attacks. In the last decades, tremendous progress has been made toward deciphering the mechanisms by which plants perceive and respond to ethylene (Benavente and Alonso, 2006;Ju et al., 2012). Studies on components of ethylene signaling have revealed a linear transduction pathway that ultimately leads to the activation of transcriptional regulators belonging to the ethylene response factor (ERF) family of transcription factors. While the upstream components of the ethylene transduction pathway are common to all ethylene responses, the apparent simplicity of the hormone signaling pathway cannot account for the wide diversity and specificity of biological responses. ERFs are one of the largest families of plant transcription factors, and in this regard, they represent a suitable step where the diversity and specificity of ethylene responses can be expressed. These downstream components of ethylene signaling are the main mediators of ethylenedependent gene transcription.Considering the importance of fleshy fruits for a healthy diet and the prominent role assigned to ethylene in the control of fruit ripening, substantial advances have been made to uncover the molecular mechanisms that control fruit development...

Abstract-This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-␤1-induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-␤1-induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-␤1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1␣ was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-␤ receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-␤1-induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart. (Circ Res. 2008;102:185-192.)

Abstract-This study tested the hypothesis that atrial natriuretic peptide has direct antihypertrophic actions on the heart by modulating expression of genes involved in cardiac hypertrophy and extracellular matrix production. Hearts of male, atrial natriuretic peptide-null and control wild-type mice that had been subjected to pressure overload after transverse aortic constriction and control unoperated hearts were weighed and subjected to microarray, Northern blot, and immunohistochemical analyses. Microarray and Northern blot analyses were used to identify genes that are regulated differentially in response to stress in the presence and absence of atrial natriuretic peptide. Immunohistochemical analysis was used to identify and localize expression of the protein products of these genes. Atrial natriuretic peptide-null mice demonstrated cardiac hypertrophy at baseline and an exaggerated hypertrophic response to transverse aortic constriction associated with increased expression of the extracellular matrix molecules periostin, osteopontin, collagen I and III, and thrombospondin, as well as the extracellular matrix regulatory proteins, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3, and the novel growth factor pleiotrophin compared with wild-type controls. These results support the hypothesis that atrial natriuretic peptide protects against pressure overload-induced cardiac hypertrophy and remodeling by negative modulation of genes involved in extracellular matrix deposition. Key Words: atrial natriuretic factor Ⅲ constriction Ⅲ aorta Ⅲ pressure overload Ⅲ hypertrophy, cardiac Ⅲ extracellular matrix Ⅲ growth substances A trial natriuretic peptide (ANP) inhibits cell growth and proliferation and induces apoptosis in a variety of cell types, including vascular smooth muscle cells (VSMCs) and cardiomyocytes. 1-3 Intracardiac ANP might also play an important autocrine/paracrine role in modulating cardiac remodeling under stress conditions and might protect against the development of pathologic cardiac hypertrophy. 4 -7 Synthesis and release of ANP in the heart are increased under stressful conditions such as pressure and volume overloadinduced pathologic cardiac hypertrophy, exercise-induced physiologic cardiac hypertrophy, heart failure, and hypoxic pulmonary hypertension. 1,2 Expression of ANP is inversely related to cardiac growth/hypertrophy. 5,8 -11 Transgenic mice overexpressing ANP have smaller hearts than do wild-type mice, and ANP gene delivery attenuates cardiac hypertrophy in spontaneously hypertensive rats. 5,7 Conversely, transgenic mice with homozygous disruption of the pro-ANP gene (Nppa -/-mice) or the natriuretic peptide receptor-A (NPR-A) gene (Npr1 -/-mice) exhibit cardiac hypertrophy at baseline that is out of proportion to the modest elevations in blood pressure (BP) observed in these models. 8 -11 Furthermore, in Npr1 -/-mice, pressure overload induced by transverse aortic constriction (TAC) results in a greater (55%) increase in left ventricular (LV) weight than in Np...

Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

BackgroundSmall number of clusters and large variation of cluster sizes commonly exist in cluster-randomized trials (CRTs) and are often the critical factors affecting the validity and efficiency of statistical analyses. F tests are commonly used in the generalized linear mixed model (GLMM) to test intervention effects in CRTs. The most challenging issue for the approximate Wald F test is the estimation of the denominator degrees of freedom (DDF). Some DDF approximation methods have been proposed, but their small sample performances in analysing binary outcomes in CRTs with few heterogeneous clusters are not well studied.MethodsThe small sample performances of five DDF approximations for the F test are compared and contrasted under CRT frameworks with simulations. Specifically, we illustrate how the intraclass correlation (ICC), sample size, and the variation of cluster sizes affect the type I error and statistical power when different DDF approximation methods in GLMM are used to test intervention effect in CRTs with binary outcomes. The results are also illustrated using a real CRT dataset.ResultsOur simulation results suggest that the Between-Within method maintains the nominal type I error rates even when the total number of clusters is as low as 10 and is robust to the variation of the cluster sizes. The Residual and Containment methods have inflated type I error rates when the cluster number is small (<30) and the inflation becomes more severe with increased variation in cluster sizes. In contrast, the Satterthwaite and Kenward-Roger methods can provide tests with very conservative type I error rates when the total cluster number is small (<30) and the conservativeness becomes more severe as variation in cluster sizes increases. Our simulations also suggest that the Between-Within method is statistically more powerful than the Satterthwaite or Kenward-Roger method in analysing CRTs with heterogeneous cluster sizes, especially when the cluster number is small.ConclusionWe conclude that the Between-Within denominator degrees of freedom approximation method for F tests should be recommended when the GLMM is used in analysing CRTs with binary outcomes and few heterogeneous clusters, due to its type I error properties and relatively higher power.

Pancreatic beta cell loss is a key factor in the pathogenesis of type 1 diabetes (T1D), but therapies to halt this process are lacking. We previously reported that the approved antihypertensive calcium-channel blocker verapamil, by decreasing the expression of thioredoxin-interacting protein, promotes the survival of insulin-producing beta cells and reverses diabetes in mouse models. To translate these findings into humans, we conducted a randomized double-blind placebo-controlled phase 2 clinical trial ( NCT02372253 ) to assess the efficacy and safety of oral verapamil added for 12 months to a standard insulin regimen in adult subjects with recent-onset T1D. Verapamil treatment, compared with placebo was well tolerated and associated with an improved mixed-meal-stimulated C-peptide area under the curve, a measure of endogenous beta cell function, at 3 and 12 months (prespecified primary endpoint), as well as with a lower increase in insulin requirements, fewer hypoglycemic events and on-target glycemic control (secondary endpoints). Thus, addition of once-daily oral verapamil may be a safe and effective novel approach to promote endogenous beta cell function and reduce insulin requirements and hypoglycemic episodes in adult individuals with recent-onset T1D.

This study tested the hypothesis that expression of the novel adhesion molecule periostin (PN) and osteopontin (OPN) is increased in lung and in isolated pulmonary arterial smooth muscle cells (PASMCs) in response to the stress of hypoxia and explored the signaling pathways involved. Adult male rats were exposed to 10% O2 for 2 wk, and growth-arrested rat PASMCs were incubated under 1% O2 for 24 h. Hypoxia increased PN and OPN mRNA expression in rat lung. In PASMCs, hypoxia increased PN but not OPN expression. The hypoxia-responsive growth factors fibroblast growth factor-1 (FGF-1) and angiotensin II (ANG II) caused dose- and time-dependent increases in PN and OPN expression in PASMCs. FGF-1-induced PN expression was blocked by the FGF-1 receptor antagonist PD-166866 and by inhibitors of phosphatidylinositol 3-kinase (PI3K) (LY-294002, wortmannin), p70S6K (rapamycin), MEK1/2 (U-0126, PD-98059), and p38MAPK (SB-203580) but not of JNK (SP-600125). ANG II-induced PN expression was blocked by the AT(1)-receptor antagonist losartan and by inhibitors of PI3K and MEK1/2. In contrast, FGF-1-induced OPN expression was blocked by inhibitors of JNK or MEK1/2 but not of PI3K, p70S6K, or p38MAPK. Activation of p70S6K and p38MAPK by anisomycin robustly stimulated PN but not OPN expression. This study is the first to demonstrate that growth factor-induced expression of PN in PASMCs is mediated through PI3K/p70S6K, Ras/MEK1/2, and Ras/p38MAPK signaling pathways, whereas the expression of OPN is mediated through Ras/MEK1/2 and Ras/JNK signaling pathways. These differences in signaling suggest that PN and OPN may play different roles in pulmonary vascular remodeling under pathophysiological conditions.