Tandem ChoRE and CCAAT Motifs and Associated Factors Regulate Txnip Expression in Response to Glucose or Adenosine-Containing Molecules

Background: Thioredoxin interacting protein (Txnip) is a multifunctional protein involved in regulation of cell cycle events and cellular metabolism. The expression of Txnip is known to be induced by glucose, adenosine-containing molecules, and other physiological cues; however, the underlying regulatory mechanisms remain elusive. Methodology/Principal Findings: In this study, using promoter reporter, electrophoresis mobility shift (EMSA), and chromatin immuno-precipitation (ChIP) assays, we have identified an additional carbohydrate response element (ChoRE) on the promoter of Txnip gene, which functions cooperatively with the earlier identified ChoRE to mediate optimal Txnip expression. However, these two ChoREs are not sufficient to mediate the induction of Txnip expression by glucose or adenosine-containing molecules; and two CCAAT boxes, both of which can recruit nuclear factor Y (NF-Y) to the Txnip promoter, are also required for the induction. Accordingly, we have found that the function of ChoREs and associated factors is contingent on tandem CCAAT boxes, in that occupancy of the Txnip promoter by NF-Y is a prerequisite for efficacious recruitment of Mondo/MLX to ChoREs under glucose stimulation. Conclusions/Significance: Our findings suggest a synergy between the tandem CCAAT and ChoRE motifs and associated NF-Y and Mondo/MLX transcription factors in enhancing transcription from the Txnip promoter. This piece of information will be helpful for future dissection of molecular mechanisms governing the transcriptional regulation of Txnip, a glucos

STORM: A General Model to Determine the Number and Adaptive Changes of Epithelial Stem Cells in Teleost, Murine and Human Intestinal Tracts

Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0–4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0–4.1 stem cells on each crypt section of murine small intestine and 1.8–3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions.MIT-Singapore AllianceNational University of Singapore. Dept. of Biological Science

Toxicology and Drug Delivery by Cucurbit[n]uril Type Molecular Containers

Many drug delivery systems are based on the ability of certain macrocyclic compounds - such as cyclodextrins (CDs) - to act as molecular containers for pharmaceutical agents in water. Indeed beta-CD and its derivatives have been widely used in the formulation of hydrophobic pharmaceuticals despite their poor abilities to act as a molecular container (e.g., weak binding (K(a)<10(4) M(-1)) and their challenges toward chemical functionalization. Cucurbit[n]urils (CB[n]) are a class of molecular containers that bind to a variety of cationic and neutral species with high affinity (K(a)>10(4) M(-1)) and therefore show great promise as a drug delivery system.In this study we investigated the toxicology, uptake, and bioactivity of two cucurbit[n]urils (CB[5] and CB[7]) and three CB[n]-type containers (Pentamer 1, methyl hexamer 2, and phenyl hexamer 3). All five containers demonstrated high cell tolerance at concentrations of up to 1 mM in cell lines originating from kidney, liver or blood tissue using assays for metabolic activity and cytotoxicity. Furthermore, the CB[7] molecular container was efficiently internalized by macrophages indicating their potential for the intracellular delivery of drugs. Bioactivity assays showed that the first-line tuberculosis drug, ethambutol, was as efficient in treating mycobacteria infected macrophages when loaded into CB[7] as when given in the unbound form. This result suggests that CB[7]-bound drug molecules can be released from the container to find their intracellular target.Our study reveals very low toxicity of five members of the cucurbit[n]uril family of nanocontainers. It demonstrates the uptake of containers by cells and intracellular release of container-loaded drugs. These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system

Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical. © 2021 The Authors. Biotechnology Journal published by Wiley-VCH Gmb

Fusion of the Endoplasmic Reticulum and Mitochondrial Outer Membrane in Rats Brown Adipose Tissue: Activation of Thermogenesis by Ca2+

Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca2+-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca2+ effect in BAT mitochondria thermogenesis. We found that Ca2+ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca2+ concentration needed for half-maximal activation varied between 0.08 and 0.11 µM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN

A Novel Whole-Cell Biocatalyst with NAD+ Regeneration for Production of Chiral Chemicals

Background: The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P) + regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes. Methodology/Principal Findings: Simultaneous overexpression of an NAD + dependent enzyme and an NAD + regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD +-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol. Conclusions/Significance: A recombinant strain, in which an NAD + regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using differen

Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo

BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA) are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA). The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms

Система мотивации педагогов в сфере среднего образования (на примере Богашевской общеобразовательной школы)

Цель работы – разработать рекомендации для повышения эффективности системы мотивации педагогов в сфере среднего образования (на примере Богашевской средней общеобразовательной школы).В процессе исследования проводилось исследование различных подходов к системе мотивации как направления управления персоналом с учетом специфики сфере среднего образования; анализировалась система мотивации педагогов начальной школы и педагогов-предметников в средней общеобразовательной школе. В результате исследования были даны рекомендации по повышению эффективности системы мотивации трудовой деятельности педагогов в МБОУ "Богашевская СОШ им. А.И.Федорова".The aim of the work is to develop recommendations for increasing the effectiveness of the system of motivation of teachers in secondary education (by the example of Bogashevskaya secondary school). During the research, various approaches to the system motivation as a direction of personnel management, taking into account the specifics of secondary education; the system of motivation of primary school teachers and subject teachers was analyzed in the secondary general education school. As a result of the research, recommendations were given to improve the effectiveness of the motivation system for the work of teachers at the MBOU "Bogashevskaya SOSH named after. A.I.Fedorov "

MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7

MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3′-untranslated region (UTR) of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378*) plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 3′-untranslated region (3′UTR) contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 3′UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 3′UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7) for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation

Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC(20)). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K(m) for MnATP is approximately 150-fold less than that for MgATP).Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases