MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.     

Pancreatic β-cell prosurvival effects of the incretin hormones involve post-translational modification of Kv2.1 delayed rectifier channels

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that exert insulinotropic and anti-apoptotic actions on pancreatic β-cells. Insulinotropic actions of the incretins involve modulation of voltage-gated potassium (Kv) channels. In multiple cell types, Kv channel activity has been implicated in cell volume changes accompanying initiation of the apoptotic program. Focusing on Kv2.1, we examined whether regulation of Kv channels in β-cells contributes to the prosurvival effects of incretins. Overexpression of Kv2.1 in INS-1 β-cells potentiated apoptosis in response to mitochondrial and ER stress and, conversely, co-stimulation with GIP/GLP-1 uncoupled this potentiation, suppressing apoptosis. In parallel, incretins promoted phosphorylation and acetylation of Kv2.1 via pathways involving protein kinase A (PKA)/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC). Further studies demonstrated that acetylation of Kv2.1 was mediated by incretin actions on nuclear/cytoplasmic shuttling of CREB binding protein (CBP) and its interaction with Kv2.1. Regulation of β-cell survival by GIP and GLP-1 therefore involves post-translational modifications (PTMs) of Kv channels by PKA/MSK-1 and HAT/HDAC. This appears to be the first demonstration of modulation of delayed rectifier Kv channels contributing to the β-cell prosurvival effects of incretins and of 7-transmembrane G protein-coupled receptor (GPCR)-stimulated export of a nuclear lysine acetyltransferase that regulates cell surface ion channel function.     

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.     

Feasibility of hydrogen production from ripened fruits by a combined two-stage (dark/dark) fermentation system

Anaerobic fermentation for hydrogen (H₂) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H₂ production (4463.7 mL-H₂ L⁻¹-culture) with a maximum H₂ yield (2.2 mol H₂ mol⁻¹ glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H₂ L⁻¹-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H₂ conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system.     

Preparation of photolithographically patterned inverse opal hydrogel microstructures and its application to protein patterning

Protein pattern has played an important role in biosensors, bioMEMS, tissue engineering, fundamental studies of cell biology, and basic proteomics research. Here, we developed a straightforward and effective protein patterning technique using macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel micropatterns as a three-dimensional (3D) template for protein immobilization. Micropatterns of macroporous hydrogels with inverse opal structures were prepared on poly(ethylene glycol) (PEG)-coated silicon substrates by combining a colloidal crystal templating method with photopatterning. The resultant inverse opal hydrogel (IOH) micropatterns were modified with 3-aminopropyltriethoxysilane using the hydroxyl groups in PHEMA for the covalent immobilization of proteins. Proteins were selectively immobilized only on the hydrogel micropatterns, while the PEG regions served as an effective barrier to protein adsorption. Because of their highly ordered and interconnected 3D macroporous structures and large internal surface areas, protein loading in the IOH micropattern was about six times greater than that on a non-porous hydrogel micropattern, which consequently improved the protein activity. The porosity of the hydrogel micropatterns could be controlled using different sizes of colloidal nanoparticles, and using smaller nanoparticles produced hydrogel micropatterns with higher protein loading capacities and activities. To demonstrate the potential use of IOH micropatterns in biosensor systems, biotin was micropatterned on the hydrogels and the specific binding of streptavidin was successfully assayed using IOH micropatterns with better fluorescence signals and sensitivity than that of the corresponding non-porous hydrogel micropatterns.     

Fabrication of degradable carboxymethyl cellulose (CMC) microneedle with laser writing and replica molding process for enhancement of transdermal drug delivery

Transdermal drug delivery system (TDDS) may provide a more reliable method of drug delivery than oral delivery by avoiding gut absorption and first-pass metabolism, but needs a method for efficiently crossing the epidermal barrier. To enhance the delivery through the skin, we have developed a biocompatible, dissolvable microneedle array made from carboxymethyl cellulose (CMC). Using laser ablation for creating the mold greatly improved the efficiency and reduced the cost of microneedle fabrication. Mixing CMC with amylopectin (AP) enhanced the mechanical and tunable dissolution properties of the microneedle for controlled release of model compounds. Using the CMC microneedle array, we observed significant enhancement in the skin permeability of a fluorescent model compound, and also increase in the anti-oxidant activity of ascorbic acid after crossing the skin. Our dissolvable microneedle array provides a new and biocompatible method for delivery of drugs and cosmetic compounds through the skin.     

Mig-6 plays a critical role in the regulation of cholesterol homeostasis and bile Acid synthesis

The disruption of cholesterol homeostasis leads to an increase in cholesterol levels which results in the development of cardiovascular disease. Mitogen Inducible Gene 6 (Mig-6) is an immediate early response gene that can be induced by various mitogens, stresses, and hormones. To identify the metabolic role of Mig-6 in the liver, we conditionally ablated Mig-6 in the liver using the Albumin-Cre mouse model (Alb(cre/+)Mig-6(f/f); Mig-6(d/d)). Mig-6(d/d) mice exhibit hepatomegaly and fatty liver. Serum levels of total, LDL, and HDL cholesterol and hepatic lipid were significantly increased in the Mig-6(d/d) mice. The daily excretion of fecal bile acids was significantly decreased in the Mig-6(d/d) mice. DNA microarray analysis of mRNA isolated from the livers of these mice showed alterations in genes that regulate lipid metabolism, bile acid, and cholesterol synthesis, while the expression of genes that regulate biliary excretion of bile acid and triglyceride synthesis showed no difference in the Mig-6(d/d) mice compared to Mig-6(f/f) controls. These results indicate that Mig-6 plays an important role in cholesterol homeostasis and bile acid synthesis. Mice with liver specific conditional ablation of Mig-6 develop hepatomegaly and increased intrahepatic lipid and provide a novel model system to investigate the genetic and molecular events involved in the regulation of cholesterol homeostasis and bile acid synthesis. Defining the molecular mechanisms by which Mig-6 regulates cholesterol homeostasis will provide new insights into the development of more effective ways for the treatment and prevention of cardiovascular disease.     

Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli

Proteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and IbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

An Imported Case of Cystic Echinococcosis in the Liver

A 25-year-old Uzbek male presented with right upper abdominal pain for 20 days. On radiologic studies, a huge cystic mass was noticed in the right liver which was suspected as parasitic. The patient received right hepatic segmentectomy (segment 7), and the surgically resected mass was confirmed as cystic echinococcosis (CE), measuring 10.5 cm in its diameter. The inner surface of the cyst was bile-stained. The patient was discharged on the 8th hospital day, and was rechecked 6 months after the surgical intervention without any evidence of recurrence. The present report describes findings of an imported case of CE which represented ultrasound images of the ''ball of wool''.