The Viking mission: Implications for life on Mars

Summary The results of the Viking Biology experiments are best explained by non-biological phenomena: The interaction of the reagents with the materials comprising the regolith. Conditions of water activity, temperature, availability of carbon sources and others in most regions of the planet are too extreme for survival and growth of any known Earth microorganisms. Although the possibility persists that some very unusual form of life is somewhere on that planet the evidence is best interpreted as negative. Even though there is no evidence for current life on Mars, whether or not life ever originated there is not known.     

Early molecular events in the interaction of enveloped viruses with cells

The fluorescence depolarization of 1,6-diphenyl-hexatriene was used to study the dynamic properties of the hydrophobic regions of the lipid envelopes of ortho- and paramyxoviruses as well as of the Rous sarcoma virus and of the membrane lipids of susceptible and nonsusceptible cells.The systems investigated where active and inactive influenza viruses, and NDV virus acting on chick embryo fibroblasts and Rous sarcoma virus acting on susceptible (C/E) and nonsusceptible (C/B) chicken-cell.Polarization degrees and mean rotational correlation times of DPH embedded in viral lipids were significantly higher than those of DPH in the cell membranes, due to a higher rigidity of the virus envelopes. When suspensions of labelled viruses and unlabelled cells or unlabelled viruses and labelled cells were mixed, a characteristic change of the fluorescence polarization degrees with time was observed. This behaviour was ascribed to a label transfer from virus to cell membranes or vice versa.While the rate constants of label transfer from virus to cells and cells to virus were about the same for the penetrating viruses the rate constants of label release from inactive virus to cells were much larger than for the migration in the opposite direction.     

Interrogation of phosphor-specific interaction on a high-throughput label-free optical biosensor system–Epic® system

The Epic^® system, a high-throughput label-free optical biosensor system, is applied for the biochemical interrogation of phosphor-specific interactions of the 14-3-3 protein and its substrates. It has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. A perspective of biochemical applications for diagnostics and biomarker discovery, as well as cell-based applications for endogenous receptors and viral infection characterization, are also provided.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

Benchmarking sample preparation/digestion protocols reveals tube‐gel being a fast and repeatable method for quantitative proteomics

Sample preparation, typically by in‐solution or in‐gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in‐gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS‐PAGE is a time‐consuming approach. Tube‐gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label‐free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label‐free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG‐prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841).     

The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).     

Label‐free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non‐MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label‐free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p‐value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS‐enriched proteins.     

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.     

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label‐free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron‐mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.