BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation

The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267–337). Deletion of a BECN1 ECD subregion (amino acids 285–299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.     

Estimating time since infection in early homogeneous HIV-1 samples using a poisson model

Background  

The occurrence of a genetic bottleneck in HIV sexual or mother-to-infant transmission has been well documented. This results in a majority of new infections being homogeneous, i.e., initiated by a single genetic strain. Early after infection, prior to the onset of the host immune response, the viral population grows exponentially. In this simple setting, an approach for estimating evolutionary and demographic parameters based on comparison of diversity measures is a feasible alternative to the existing Bayesian methods (e.g., BEAST), which are instead based on the simulation of genealogies.     

A novel type-III staphylococcal cassette chromosome mec (SCCmec) variant among Indian isolates of methicillin-resistant Staphylococcus aureus

We identified a novel type-III staphylococcal cassette chromosome mec (SCC mec ) element carried by eight methicillin-resistant Staphylococcus aureus (MRSA) strains from different wards and patients in an Indian hospital. Although the pulsed-field gel electrophoresis pattern and spa types of eight strains were identical and clonally related to other nosocomial Indian isolates that belonged to sequence type (ST) 239 and spa type t037, the minimum inhibitory concentration (MIC) of these eight variants was noticeably low compared with the typical type-III isolates from the same hospital, and we were unable to identify ccrC and hsdR by multiplex PCR, although mer operon and transposases A, B, and C of Tn 554 were amplified. By amplifying the entire SCC mec region by long-range PCR and determining parts of the nucleotide sequences of one isolate (V14), we found that the strain carried a novel SCC mec element containing a 422 bp sequence, which is highly homologous to that identified in strain CCR1-9583, mer operon and plasmid pT181 integrated in tandem via IS 431 in the J3 region. It also carried a cassette chromosome, previously reported to be an SCC-like element, downstream of type-III SCC mec . Because PCR amplification of representative genes showed that these eight strains carried the same genetic elements, they belong to a novel MRSA clone that differs from most nosocomial clones carrying type-III SCC mec and SCC mercury , despite belonging to the ST239 genotype.     

Hydrogen Photoproduction by Immobilized N2-Fixing Cyanobacteria: Understanding the Role of the Uptake Hydrogenase in the Long-Term Process

We have investigated two approaches to enhance and extend H₂ photoproduction yields in heterocystous, N₂-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO₂ supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H₂ production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N₂ by periodic treatments of air, or air plus CO₂. These treatments restored the photosynthetic activity of the cells and led to a high level of H₂ production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO₂) but not in the Anabaena PCC 7120 strain (for which H₂ yields did not change after air treatments). The highest H₂ yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO₂ under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O₂ from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.     

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7 d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1 mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1 mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.     

Heterologous Expression of Oxalate Decarboxylase in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> NC8

Lactic acid bacteria (LABs) are being used as a probiotic very often for various enteric problems. Many genetically modified LABs are created by different workers for various novel applications. In this study we examine the expression of heterologous oxalate decarboxylase (oxdc) in Lactobacillus plantarum NC8. Generally, this enzyme is not present in Lactobacillus spp. Oxdc gene from Bacillus subtilis was polymerase chain reaction-amplified and cloned in a shuttle vector pSIP400 series, downstream of the inducible promoter, P_orfx. In the presence of an inducing peptide, Sakacin-P, the expression of OxdC was observed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cell-free extract and the purified protein from the recombinant LABs showed the presence of OxdC activity. The above recombinant LABs, with desired modifications, can be used as a possible probiotic for the degradation of intestinal dietary oxalate for preventing enteric hyperoxaluria.     

Sigma-2: Multiple sequence alignment of non-coding DNA via an evolutionary model

Background  

While most multiple sequence alignment programs expect that all or most of their input is known to be homologous, and penalise insertions and deletions, this is not a reasonable assumption for non-coding DNA, which is much less strongly conserved than protein-coding genes. Arguing that the goal of sequence alignment should be the detection of homology and not similarity, we incorporate an evolutionary model into a previously published multiple sequence alignment program for non-coding DNA, Sigma, as a sensitive likelihood-based way to assess the significance of alignments. Version 1 of Sigma was successful in eliminating spurious alignments but exhibited relatively poor sensitivity on synthetic data. Sigma 1 used a p-value (the probability under the "null hypothesis" of non-homology) to assess the significance of alignments, and, optionally, a background model that captured short-range genomic correlations. Sigma version 2, described here, retains these features, but calculates the p-value using a sophisticated evolutionary model that we describe here, and also allows for a transition matrix for different substitution rates from and to different nucleotides. Our evolutionary model takes separate account of mutation and fixation, and can be extended to allow for locally differing functional constraints on sequence.