Robust inference for the stepped wedge design

Stepped wedge designed trials are a type of cluster-randomized study in which the intervention is introduced to each cluster in a random order over time. This design is often used to assess the effect of a new intervention as it is rolled out across a series of clinics or communities. Based on a permutation argument, we derive a closed-form expression for an estimate of the intervention effect, along with its standard error, for a stepped wedge design trial. We show that these estimates are robust to misspecification of both the mean and covariance structure of the underlying data-generating mechanism, thereby providing a robust approach to inference for the intervention effect in stepped wedge designs. We use simulations to evaluate the type 1 error and power of the proposed estimate and to compare the performance of the proposed estimate to the optimal estimate when the correct model specification is known. The limitations, possible extensions, and open problems regarding the method are discussed.     

Melatonin protects against defects induced by malathion during porcine oocyte maturation

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.     

Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling

The motility of mesenchymal stem cells (MSCs) is highly related to their homing in vivo, a critical issue in regenerative medicine. Our previous study indicated copper (Cu) might promote the recruitment of endogenous MSCs in canine esophagus defect model. In this study, we investigated the effect of Cu on the motility of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism in vitro. Cu supplementation could enhance the motility of BMSCs, and upregulate the expression of hypoxia-inducible factor 1α (Hif1α) at the protein level, and upregulate the expression of rho family GTPase 3 (Rnd3) at messenger RNA and protein level. When Hif1α was silenced by small interfering RNA (siRNA), Cu-induced Rnd3 upregulation was blocked. When Rnd3 was silenced by siRNA, the motility of BMSCs was decreased with or without Cu supplementation, and Cu-induced cytoskeleton remodeling was neutralized. Furthermore, overexpression of Rnd3 also increased the motility of BMSCs and induced cytoskeleton remodeling. Overall, our results demonstrated that Cu enhanced BMSCs migration through, at least in part, cytoskeleton remodeling via Hif1α-dependent upregulation of Rnd3. This study provided an insight into the mechanism of the effect of Cu on the motility of BMSCs, and a theoretical foundation of applying Cu to improve the recruitment of BMSCs in tissue engineering and cytotherapy.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Cloning and evolutionary analysis of tobacco MAPK gene family

The mitogen-activated protein (MAP) kinase cascade is an important signaling module which is involved in biotic and abiotic stress responses as well as plant growth and development. In this study, we identified 17 tobacco MAPKs including 11 novel tobacco MAPK genes that have not been identified before. Comparative analysis with MAPK gene families from other plants, such as Athaliana thaliana, rice and poplar, suggested that tobacco MAPKs (such as NtMPK1, NtMPK3 and NtMPK8) might play similar functions in response to abiotic and biotic stresses. QRT-PCR analysis revealed that a total of 14 NtMPKs were regulated by SA and/or MeJA, suggesting their potential roles involved in plant defense response. In addition, 6 NtMPKs were induced by drought treatment, implying their roles in response to drought stress. Our results indicated that most of tobacco MAPK might be involved in plant defense response, which provides the basis for further analysis on physiological functions of tobacco MAPKs.     

Neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside against sodium nitroprusside-induced neurotoxicity in HT22 cells

In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.