Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKKβ

We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca²⁺-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.     

Silk fibroin hydrogels for potential applications in photodynamic therapy

In this study, we prepared translucid hydrogels with different concentrations of silk fibroin, extracted from raw silk fibers, and used them as a matrix to incorporate the photosensitizer 5-(4-aminophenyl)-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium for application in photodynamic therapy (PDT). The hydrogels obtained were characterized by rheology, spectrophotometry, and scattering techniques to elucidate the factors involved in the formation of the hydrogel, and to characterize the behavior of silk fibroin (SF) after incorporating of the porphyrin to the matrix. The rheology results demonstrated that the SF hydrogels had a shear thinning behavior. In addition, we were able to verify that the structure of the material was able to be recovered over time after shear deformation. The encapsulation of porphyrins in hydrogels leads to the formation of self-assembled peptide nanostructures that prevent porphyrin aggregation, thereby greatly increasing the generation of singlet oxygen. Also, our findings suggest that porphyrin can diffuse out of the hydrogel and permeate the outer skin layers. This evidence suggests that SF hydrogels could be used as porphyrin encapsulation and as a drug carrier for the sustained release of photosensitizers for PDT.     

AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable alphabetagamma heterotrimer comprising a catalytic alpha and two non-catalytic subunits, beta and gamma. The beta subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the beta subunit, beta1-(186-270), is sufficient to form an active AMP-dependent heterotrimer alpha1beta1-(186-270)-gamma1, whereas the 25-residue beta1 C-terminal (246-270) sequence is sufficient to bind gamma1, gamma2, or gamma3 but not the alpha subunit. Deletion of the beta C-terminal Ile-270 precludes betagamma association in the absence of the alpha subunit, but the presence of the alpha subunit or substitution of Ile-270 with Ala or Glu restores betagamma binding. Truncation of the alpha subunit reveals that beta1 binding requires the alpha1-(313-473) sequence. The conserved C-terminal 85-residue sequence of the beta subunit (90% between beta1 and beta2) is the primary alphagamma binding sequence responsible for the formation of the AMPK alphabetagamma heterotrimer.     

UV-Light Effects on Cytochrome C Modulated by the Aggregation State of Phenothiazines

The present study shows the factors that modulate the photodamage promoted by phenothiazines. Cytochrome c was irradiated with UV light for 120 min, over a pH range from 4.0 to 8.0, in the absence and in the presence of different concentrations of thioridazine (TR) and fluphenazine (FP). In the absence of phenothiazines, the maximal rate of a Soret band blue shift (nm/min) from 409 to 406 nm was obtained at pH 4.0 (0.028 nm/min). The presence of phenothiazines at the concentration range 10-25 µmol/L amplified and accelerated a cytochrome c blue shift (409 to 405 nm, at a rate = 0.041 nm/min). Above 25 µmol/L, crescent concentrations of phenothiazines contributed to cytochrome c protection with (maximal at 2500 µmol/L). Scanning electronic microscopy revealed the formation of nanostructures. The pH also influenced the effect of low phenothiazine concentrations on cytochrome c. Thus, the predominance of phenothiazine-promoted cytochrome c damage or protection depends on a balance of the following factors: the yield of photo-generated drug cation radicals, which is favored by acidic pH; the stability of the cation radicals, which is favored by the drug aggregation; and the cytochrome c structure, modulated by the pH.     

The role of AdipoR1 and AdipoR2 in liver fibrosis

Activation of the adiponectin (APN) signaling axis retards liver fibrosis. However, understanding of the role of AdipoR1 and AdipoR2 in mediating this response is still rudimentary. Here, we sought to elucidate the APN receptor responsible for limiting liver fibrosis by employing AdipoR1 and AdipoR2 knock-out mice in the carbon tetrachloride (CCl₄) model of liver fibrosis. In addition, we knocked down receptor function in primary hepatic stellate cells (HSCs) in vitro. Following the development of fibrosis, AdipoR1 and AdipoR2 KO mice had no quantitative difference in fibrosis by Sirius red staining. However, AdipoR2 KO mice had an enhanced fibrotic signature with increased Col1-α1, TGFß-1, TIMP-1, IL-10, MMP-2 and MMP-9. Knockdown of AdipoR1 or AdipoR2 in HSCs followed by APN treatment demonstrated that AdipoR1 and AdipoR2 did not affect proliferation or TIMP-1 gene expression, while AdipoR2 modulated Col1-α1 and α-SMA gene expression, HSC migration, and AMPK activity. These finding suggest that AdipoR2 is the major APN receptor on HSCs responsible for mediating its anti-fibrotic effects.     

Taxonomy‐free molecular diatom index for high‐throughput eDNA biomonitoring

Current biodiversity assessment and biomonitoring are largely based on the morphological identification of selected bioindicator taxa. Recently, several attempts have been made to use eDNA metabarcoding as an alternative tool. However, until now, most applied metabarcoding studies have been based on the taxonomic assignment of sequences that provides reference to morphospecies ecology. Usually, only a small portion of metabarcoding data can be used due to a limited reference database and a lack of phylogenetic resolution. Here, we investigate the possibility to overcome these limitations using a taxonomy‐free approach that allows the computing of a molecular index directly from eDNA data without any reference to morphotaxonomy. As a case study, we use the benthic diatoms index, commonly used for monitoring the biological quality of rivers and streams. We analysed 87 epilithic samples from Swiss rivers, the ecological status of which was established based on the microscopic identification of diatom species. We compared the diatom index derived from eDNA data obtained with or without taxonomic assignment. Our taxonomy‐free approach yields promising results by providing a correct assessment for 77% of examined sites. The main advantage of this method is that almost 95% of OTUs could be used for index calculation, compared to 35% in the case of the taxonomic assignment approach. Its main limitations are under‐sampling and the need to calibrate the index based on the microscopic assessment of diatoms communities. However, once calibrated, the taxonomy‐free molecular index can be easily standardized and applied in routine biomonitoring, as a complementary tool allowing fast and cost‐effective assessment of the biological quality of watercourses.     

Probabilistic base calling of Solexa sequencing data

Background  

Solexa/Illumina short-read ultra-high throughput DNA sequencing technology produces millions of short tags (up to 36 bases) by parallel sequencing-by-synthesis of DNA colonies. The processing and statistical analysis of such high-throughput data poses new challenges; currently a fair proportion of the tags are routinely discarded due to an inability to match them to a reference sequence, thereby reducing the effective throughput of the technology.     

MAR‐Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.     

The evolution of insulin resistance in muscle of the glucose infused rat

Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser⁴⁷³) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser⁹) and AS160 (Thr⁶⁴²) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.