Advancements in the Analysis of the Arabidopsis Plasma Membrane Proteome

The plasma membrane (PM) regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. PM microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed

Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection (c) occupational exposure in a wool-sorters factory (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups.Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However a number of other chromosomally-located and plasmid encoded open reading frames were also recognised by infected or exposed groups in comparison to controls. Some of these antigens e.g. BA4182 are not recognised by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo and are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis ‘infectome’. These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesised, tested in mouse immunogenicity studies and validated in parallel using human sera from the same study

Lysosomal dysfunction in muscle with special reference to glycogen storage disease type II

The importance of proper lysosomal activity in cell and tissue homeostasis is underlined by experiments of nature , i.e. genetic defects in one of the at least 40 lysosomal enzymes/proteins present in the human cell. The complete lack of 1-4 a-glucosidase (glycogen storage disease type II (GSD II) or Pompe disease) is life-threatening. Patients suffering from GSD II commonly die before the age of 2 years because of cardiorespiratory insufficiency. Striated muscle cells appear to be particularly vulnerable in GSD II. The high cytoplasmic glycogen content in muscle cells most likely gives rise to a high rate of glycogen engulfment by the lysosomes. The polysaccharides become subsequently trapped in these organelles when 1-4 a-glucosidase activity is absent. During the course of the disease, muscle wasting occurs. It is hypothesised that the gradual loss of muscle mass is caused by a combination of disuse atrophy and lipofuscine-mediated apoptosis of myocytes. Moreover, we hypothesise that in the remaining skeletal muscle cells, longitudinal transmission of force is hampered by swollen lysosomes, clustering of non-contractile material and focal regions with degraded contractile proteins, which results in muscle weakness

Accurate whole human genome sequencing using reversible terminator chemistry

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications