Tetrandrine blocks autophagic flux and induces apoptosis via energetic impairment in cancer cells

Lysosomes are acidic organelles that have a crucial role in degrading intracellular macromolecules and organelles during the final stage of autophagy. Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, was reported as an autophagy activator. Here, in contrast with previous studies, we show that Tet is a potent lysosomal deacidification agent and is able to block autophagic flux in the degradation stage. Single-agent Tet induces significant apoptosis both in vitro and in xenograft models. In the presence of Tet, apoptosis was preceded by a robust accumulation of autophagosomes and an increased level of microtubule-associated protein 1 light chain 3, type II (LC3-II). However, Tet increased the level of sequestosome 1 and decreased the turnover of LC3, indicating the blockade of autophagic flux in the degradation stage. As blockade of autophagic flux decreases the recycling of cellular fuels, Tet reduces the uptake of glucose in cancer cells. These effects lead to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. Blunting autophagosome formation using 3-methyladenine or genetic knockdown of Beclin-1 failed to rescue cells upon Tet treatment. By contrast, addition of methyl pyruvate to supplement TCA substrates protected Tet-treated tumor cells. These results demonstrate that energetic impairment is required in Tet-induced apoptosis. Tet, as a potent lysosomal inhibitor, is translatable to the treatment of malignant tumor patients.     

Increasing the complexity: new genes and new types of albinism

Albinism is a rare genetic condition globally characterized by a number of specific deficits in the visual system, resulting in poor vision, in association with a variable hypopigmentation phenotype. This lack or reduction in pigment might affect the eyes, skin, and hair (oculocutaneous albinism, OCA), or only the eyes (ocular albinism, OA). In addition, there are several syndromic forms of albinism (e.g. Hermansky–Pudlak and Chediak–Higashi syndromes, HPS and CHS, respectively) in which the described hypopigmented and visual phenotypes coexist with more severe pathological alterations. Recently, a locus has been mapped to the 4q24 human chromosomal region and thus represents an additional genetic cause of OCA, termed OCA5, while the gene is eventually identified. In addition, two new genes have been identified as causing OCA when mutated: SLC24A5 and C10orf11, and hence designated as OCA6 and OCA7, respectively. This consensus review, involving all laboratories that have reported these new genes, aims to update and agree upon the current gene nomenclature and types of albinism, while providing additional insights from the function of these new genes in pigment cells.     

Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10⁴ cells/mL (8.9×10³ cells/cm²) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).     

Complete structure of nuclear rDNA of the obligate plant parasite Plasmodiophora brassicae: intraspecific polymorphisms in the exon and group I intron of the large subunit rDNA

Plasmodiophora brassicae is a soil-borne obligate intracellular parasite in the phylum Cercozoa of the Rhizaria that causes clubroot disease of crucifer crops. To control the disease, understanding the distribution and infection routes of the pathogen is essential, and thus development of reliable molecular markers to discriminate geographic populations is required. In this study, the nuclear ribosomal RNA gene (rDNA) repeat unit of P. brassicae was determined, with particular emphasis on the structure of large subunit (LSU) rDNA, in which polymorphic regions were expected to be present. The complete rDNA complex was 9513bp long, which included the small subunit, 5.8S and LSU rDNAs as well as the internal transcribed spacer and intergenic spacer regions. Among eight field populations collected from throughout Honshu Island, Japan, a 1.1 kbp region of the LSU rDNA, including the divergent 8 domain, exhibited intraspecific polymorphisms that reflected geographic isolation of the populations. Two new group I introns were found in this region in six out of the eight populations, and the sequences also reflected their geographic isolation. The polymorphic region found in this study may have potential for the development of molecular markers for discrimination of field populations/isolates of this organism.     

Alfy-dependent elimination of aggregated proteins by macroautophagy: Can there be too much of a good thing?

Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to nonselective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.Key words: autophagy, protein aggregates, neurodegeneration, Alfy, aggregation, degradation     

The relationship between psychosocial stress, age, BMI, CRP, lifestyle, and the metabolic syndrome in apparently healthy subjects

The aim of this cross-sectional study was to examine the factors which may be associated with the metabolic syndrome by exploring the relationship between psychosocial stress, age, body mass index (BMI), C-reactive protein (CRP), lifestyle factors, and the components of the metabolic syndrome, such as glycated hemoglobin (HbA1c), fasting blood sugar (FBS), body fat percentage, and triglyceride concentration, among apparently healthy subjects. Psychosocial stress was measured by the use of the inventory to measure psychosocial stress (IMPS). One thousand four hundred and ninety-nine people out of 1,941 public school workers admitted to a hospital for a medical check-up responded to the IMPS, yielding a response rate of 77.2%. A total of 1,201 workers excluding 298 who were taking medication for various diseases were analyzed with the use of hierarchical multiple regression models. It was found that IMPS-measured stress score, age, BMI, and smoking habit were associated with an increase in glycated hemoglobin among men, while alcohol consumption was associated with a decrease in glycated hemoglobin. Stress score, age, BMI, and alcohol consumption were found to be associated with an increase in FBS among men, while smoking and exercise habits were associated with a decrease in FBS. CRP was found to be associated with an increase in body fat percentage among men, though stress score was not associated with an increase in body fat percentage. Stress score, age, and BMI were associated with an increase in triglyceride concentration among women. The findings of the present study seem to be in line with the hypothesis that psychosocial stress plays an important role in developing the metabolic syndrome, which may be associated with inflammatory processes in the vascular wall, resulting in atherosclerosis and cardiovascular disease.     

Long astral microtubules and RACK-1 stabilize polarity domains during maintenance phase in Caenorhabditis elegans embryos

Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity.     

Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity.     

Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXα revealed by phage display

4Z,15Z-bilirubin-IXα (4Z,15Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobilized 4Z,15Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer (P-form) of 4Z,15Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4Z,15Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4Z,15Z-BR showed higher affinity for domain I than for domain II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR.     

Causal graph-based analysis of genome-wide association data in rheumatoid arthritis

Background

GWAS owe their popularity to the expectation that they will make a major impact on diagnosis, prognosis and management of disease by uncovering genetics underlying clinical phenotypes. The dominant paradigm in GWAS data analysis so far consists of extensive reliance on methods that emphasize contribution of individual SNPs to statistical association with phenotypes. Multivariate methods, however, can extract more information by considering associations of multiple SNPs simultaneously. Recent advances in other genomics domains pinpoint multivariate causal graph-based inference as a promising principled analysis framework for high-throughput data. Designed to discover biomarkers in the local causal pathway of the phenotype, these methods lead to accurate and highly parsimonious multivariate predictive models. In this paper, we investigate the applicability of causal graph-based method TIE* to analysis of GWAS data. To test the utility of TIE*, we focus on anti-CCP positive rheumatoid arthritis (RA) GWAS datasets, where there is a general consensus in the community about the major genetic determinants of the disease.

Results

Application of TIE* to the North American Rheumatoid Arthritis Cohort (NARAC) GWAS data results in six SNPs, mostly from the MHC locus. Using these SNPs we develop two predictive models that can classify cases and disease-free controls with an accuracy of 0.81 area under the ROC curve, as verified in independent testing data from the same cohort. The predictive performance of these models generalizes reasonably well to Swedish subjects from the closely related but not identical Epidemiological Investigation of Rheumatoid Arthritis (EIRA) cohort with 0.71-0.78 area under the ROC curve. Moreover, the SNPs identified by the TIE* method render many other previously known SNP associations conditionally independent of the phenotype.

Conclusions

Our experiments demonstrate that application of TIE* captures maximum amount of genetic information about RA in the data and recapitulates the major consensus findings about the genetic factors of this disease. In addition, TIE* yields reproducible markers and signatures of RA. This suggests that principled multivariate causal and predictive framework for GWAS analysis empowers the community with a new tool for high-quality and more efficient discovery.

Reviewers

This article was reviewed by Prof. Anthony Almudevar, Dr. Eugene V. Koonin, and Prof. Marianthi Markatou.