Milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins and medium to low abundance whey proteins such as ß-lactoglobulin, lactoferrin, immunoglobulins, glycoproteins, peptide hormones, and enzymes. A sample preparation method that enables high reproducibility and throughput is key in reliably identifying proteins present or proteins responding to conditions such as a diet, health or genetics. Using skim milk samples from Jersey and Holstein-Friesian cows, we compared three extraction procedures which have not previously been applied to samples of cows' milk. Method A (urea) involved a simple dilution of the milk in a urea-based buffer, method B (TCA/acetone) involved a trichloroacetic acid (TCA)/acetone precipitation, and method C (methanol/chloroform) involved a tri-phasic partition method in chloroform/methanol solution. Protein assays, SDS-PAGE profiling, and trypsin digestion followed by nanoHPLC-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analyses were performed to assess their efficiency. Replicates were used at each analytical step (extraction, digestion, injection) to assess reproducibility. Mass spectrometry (MS) data are available via ProteomeXchange with identifier PXD002529. Overall 186 unique accessions, major and minor proteins, were identified with a combination of methods. Method C (methanol/chloroform) yielded the best resolved SDS-patterns and highest protein recovery rates, method A (urea) yielded the greatest number of accessions, and, of the three procedures, method B (TCA/acetone) was the least compatible of all with a wide range of downstream analytical procedures. Our results also highlighted breed differences between the proteins in milk of Jersey and Holstein-Friesian cows.

Several cyanobacterial species are dominant primary producers in hot spring microbial mats. To date, hot spring cyanobacterial taxonomy, as well as the evolution of their genomic adaptations to high temperatures, are poorly understood, with genomic information currently available for only a few dominant genera, including Fischerella and Synechococcus. To address this knowledge gap, the present study expands the genomic landscape of hot spring cyanobacteria and traces the phylum-wide genomic consequences of evolution in high temperature environments. From 21 globally distributed hot spring metagenomes, with temperatures between 32 and 75°C, 57 medium- and high-quality cyanobacterial metagenome-assembled genomes were recovered, representing taxonomic novelty for 1 order, 3 families, 15 genera and 36 species. Comparative genomics of 93 hot spring genomes (including the 57 metagenome-assembled genomes) and 66 non-thermal genomes, showed that the former have smaller genomes and a higher GC content, as well as shorter proteins that are more hydrophilic and basic, when compared to the non-thermal genomes. Additionally, the core accessory orthogroups from the hot spring genomes of some genera had a greater abundance of functional categories, such as inorganic ion metabolism, translation and post-translational modifications. Moreover, hot spring genomes showed increased abundances of inorganic ion transport and amino acid metabolism, as well as less replication and transcription functions in the protein coding sequences. Furthermore, they showed a higher dependence on the CRISPR-Cas defense system against exogenous nucleic acids, and a reduction in secondary metabolism biosynthetic gene clusters. This suggests differences in the cyanobacterial response to environment-specific microbial communities. This phylum-wide study provides new insights into cyanobacterial genomic adaptations to a specific niche where they are dominant, which could be essential to trace bacterial evolution pathways in a warmer world, such as the current global warming scenario.

Chronic obstructive pulmonary disease (COPD) is a complex and heritable disease, associated with multiple genetic variants. Specific familial types of COPD may be explained by rare variants, which have not been widely studied. We aimed to discover rare genetic variants underlying COPD through a genome-wide linkage scan. Affected-only analysis was performed using the 6K Illumina Linkage IV Panel in 142 cases clustered in 27 families from a genetic isolate, the Erasmus Rucphen Family (ERF) study. Potential causal variants were identified by searching for shared rare variants in the exome-sequence data of the affected members of the families contributing most to the linkage peak. The identified rare variants were then tested for association with COPD in a large meta-analysis of several cohorts. Significant evidence for linkage was observed on chromosomes 15q14-15q25 [logarithm of the odds (LOD) score = 5.52], 11p15.4-11q14.1 (LOD = 3.71) and 5q14.3-5q33.2 (LOD = 3.49). In the chromosome 15 peak, that harbors the known COPD locus for nicotinic receptors, and in the chromosome 5 peak we could not identify shared variants. In the chromosome 11 locus, we identified four rare (minor allele frequency (MAF) <0.02), predicted pathogenic, missense variants. These were shared among the affected family members. The identified variants localize to genes including neuroblast differentiation-associated protein (AHNAK), previously associated with blood biomarkers in COPD, phospholipase C Beta 3 (PLCB3), shown to increase airway hyper-responsiveness, solute carrier family 22-A11 (SLC22A11), involved in amino acid metabolism and ion transport, and metallothionein-like protein 5 (MTL5), involved in nicotinate and nicotinamide metabolism. Association of SLC22A11 and MTL5 variants were confirmed in the meta-analysis of 9,888 cases and 27,060 controls. In conclusion, we have identified novel rare variants in plausible genes related to COPD. Further studies utilizing large sample whole-genome sequencing should further confirm the associations at chromosome 11 and investigate the chromosome 15 and 5 linked regions.

Amaryllidaceae is a large family with more than 1,600 species, belonging to 75 genera. The largest genus-Allium-is vast, comprising about a thousand species. Allium species (as well as other members of the Amaryllidaceae) are widespread and diversified, they are adapted to a wide range of habitats from shady forests to open habitats like meadows, steppes, and deserts. The genes present in chloroplast genomes (plastomes) play fundamental roles for the photosynthetic plants. Plastome traits could thus be associated with geophysical abiotic characteristics of habitats. Most chloroplast genes are highly conserved and are used as phylogenetic markers for many families of vascular plants. Nevertheless, some studies revealed signatures of positive selection in chloroplast genes of many plant families including Amaryllidaceae. We have sequenced plastomes of the following nine Allium (tribe Allieae of Allioideae) species: A. zebdanense, A. moly, A. victorialis, A. macleanii, A. nutans, A. obliquum, A. schoenoprasum, A. pskemense, A. platyspathum, A. fistulosum, A. semenovii, and Nothoscordum bivalve (tribe Leucocoryneae of Allioideae). We compared our data with previously published plastomes and provided our interpretation of Allium plastome genes' annotations because we found some noteworthy inconsistencies with annotations previously reported. For Allium species we estimated the integral evolutionary rate, counted SNPs and indels per nucleotide position as well as compared pseudogenization events in species of three main phylogenetic lines of genus Allium to estimate whether they are potentially important for plant physiology or just follow the phylogenetic pattern. During examination of the 38 species of Allium and the 11 of other Amaryllidaceae species we found that rps16, rps2, infA, ccsA genes have lost their functionality multiple times in different species (regularly evolutionary events), while the pseudogenization of other genes was stochastic events. We found that the "normal" or "pseudo" state of rps16, rps2, infA, ccsA genes correlates well with the evolutionary line of genus the species belongs to. The positive selection in various NADH dehydrogenase (ndh) genes as well as in matK, accD, and some others were found. Taking into account known mechanisms of coping with excessive light by cyclic electron transport, we can hypothesize that adaptive evolution in genes, coding subunits of NADH-plastoquinone oxidoreductase could be driven by abiotic factors of alpine habitats, especially by intensive light and UV radiation.