Cell-Autonomous Death of Cerebellar Purkinje Neurons with Autophagy in Niemann-Pick Type C Disease

Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death

Silicon modulates root phenomics and leaf ionomics in oak under Phytophthora infection and low phosphorus conditions

Pedunculate oak (Quercus robur L.) is the most abundant deciduous tree species in Europe with high economical and ecological importance. Different species of Phytophthora are considered as one of the most important factors responsible for deterioration of oak forest, causing serious root damage not only in the forest trees, but also in the nurseries. Oak seedlings were grown in plastic pots with extremely low phosphorus (P) soil (1.5 mg kg-1 total P; no available Olsen-P detected). Silicon (Si) and P were supplied as Na2SiO3 (300 mg Si kg-1 dry soil) and KH2PO4 (180 mg P kg-1 dry soil), respectively. Four treatments (-P/-Si, -P+/Si, +P/-Si, and +P/+Si) were used in the experiment. After two months of experiment, a half of the plants in each treatment were root-inoculated with Phytophthora plurivora. After further four weeks, the first symptoms of P. plurivora infection appeared in leaves (e.g., leaf necrosis and wilting). Plants were then carefully removed from the pots, divided into roots and shoots, and the roots were scanned and analyzed by the WinRHIZO® software. Foliar concentrations of Si, P, K, Ca, Mg, B, Cu, Fe, Mn, and Zn were determined by ICP-OES, while the concentrations of N and S were determined by CHNS Analyzer. The addition of Si obviously improved root health status (e.g., decreasing de number of lesions and necrosis intensity) in the infected plants grown under -P conditions, which was followed by an increased foliar P concentration. The Si supply significantly increased the root variables (e.g., total root volume, root length, and area of thin roots) in both -P and +P plants inoculated with P. plurivora. Therefore, P. plurivora infection and supply of P and Si modulated the nutrient uptake and thereby changed the leaf ionomics, especially for infected -P plants supplied with Si (e.g., significantly increased B, Cu, and Si foliar concentrations and decreased Fe, Mn, Ca, Mg, K, and S foliar concentrations). Furthermore, Si fertilization significantly declined loses in plant dry biomass caused by P. plurivora infection and/or P deficiency, showing biomass comparable to non-infected +P plants

Polymorphic expression of glutathione transferases A1, M1, P1 and T1 in epithelial ovarian cancer: a Serbian case-control study

PURPOSE: Since several studies have proposed that epithelial ovarian cancer should not be considered as a single disease entity and that it results from an accumulation of genetic changes, we aimed to assess the polymorphic expression of major cytosolic glutathione S-transferases (GSTM1, T1, A1 and P1) with respect to ovarian cancer susceptibility and aggressiveness. METHODS: This case-control study was conducted on 93 newly diagnosed epithelial ovarian cancer patients and 178 healthy matched controls. The multiplex polymerase chain reaction (PCR) was used to detect homozygous deletions of GSTM1 and GSTT1 genes. Analysis of the single nucleotide polymorphism (SNP) GSTA1 C69T was performed using PCR-restriction fragment length polymorphism (RFLP), while for SNP GSTP1 Ile105Val real-time PCR was used. RESULTS: No significant association to ovarian cancer risk was found for individual GSTM1, GSTA1 and GSTP1 genotypes (p>0.05). However, the carriers of GSTT1-active genotype were at 2-fold higher risk of ovarian cancer development (95%CI: 1.00-4.01, p=0.049), which was even more elevated in the subgroup of patients with positive family history of cancer. Moreover, the frequency of all three GST genotypes that might be associated to ovarian cancer risk (GSTT1-active, GSTA1-active and GSTP1-referent) was significantly higher in patients than in the control group (p=0.042). Even more, patients who were carriers of combination of these three genotypes represented over 64% of the total number of patients within any of the International Federation of Gynecology and Obstetrics (FIGO) stages of ovarian cancer. CONCLUSIONS: This study provides supportive evidence that GSTs might affect both susceptibility and progression of ovarian cance

Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium

A distinct pathway for ligand-stimulated lateral trafficking of the hedgehog protein Smoothened into cilia is described

A Rapid and Simple Method for DNA Engineering Using Cycled Ligation Assembly

<div><p>DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method’s utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.</p></div

DNA Fragments Used in Cycled Ligation Assembly.

<p>DNA fragments amplified from specific protein-encoding domains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-g003" target="_blank">Figure 3A</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-g003" target="_blank">Figures 3B, 3C</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-g004" target="_blank">4</a>.</p><p>DNA Fragments Used in Cycled Ligation Assembly.</p

Cycled Ligation Enables Both Complex and Multiplexed Assemblies.

<p>(<b>A</b>) Complex assembly of very small (80 bp) and large (2100 bp) DNA fragments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-t001" target="_blank"><b>Table 1</b></a>) into a single product. Six DNA fragments of various sizes were seamlessly assembled in a single reaction and amplified by PCR. (<b>B</b>) Sixteen 100–200 bp DNA sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone-0107329-t001" target="_blank"><b>Table 1</b></a>) were placed in a single cycled ligation reaction. Twelve SOCs were added to guide the assembly of four different products consisting of four inserts each (three SOCs per product). Each of the four products was PCR amplified from the single cycled ligation reaction, demonstrating that multiplexed assembly reactions work well. (<b>C</b>) Assembly of sixteen distinct DNA fragments into a single ordered product in a single reaction. The number of inserts (n) assembled into each product is displayed above lanes. All sequences, primers, and SOCs described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone.0107329.s001" target="_blank">Tables S1</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107329#pone.0107329.s002" target="_blank">S2</a></b>.</p