Proteomic analysis of hypoxia and non-hypoxia secretome mesenchymal stem-like cells from human breastmilk

IntroductionBreastmilk contains proteins and cells which have stem cell properties. The human breastmilk stem cell mimick mesenchymal stem cells and expresses pluripotency genes. The protein level of breastmilk is high in colostrum and gradually subsides in the first year of lactation. The mesenchymal stem cells from breastmilk can be an alternative source of stem cells that can potentially affect cardiovascular therapy. This study aimed to identify the proteomic analysis of secretome mesenchymal stem-like cells under hypoxia compared to non-hypoxia from human breastmilk stem cells.Material and methodsThe human breastmilk was collected from six healthy breastfeeding women and transported to the laboratory under aseptic conditions. The breastmilk cells were isolated then cultured. After 72 h, the human breastmilk stem cells reached confluence then cleaned up and isolated in serum-free media (spheroid) to allow serial passaging every 48 h. The acquisition stem cell was made with flow cytometry. The cells were divided into hBSC secretomes under hypoxia (A) and non-hypoxia (B) and analyzed for LC-MS to identify the peptide structure.ResultsThe human breastmilk cells contained several mesenchymal stem-like cells in density 2.4 × 10⁶ cell/mL for hypoxia and 2 × 10⁶ cell/mL for non-hypoxia conditions. The human breastmilk stem cell surface markers derived from the third cell passage process were 93.77% for CD44, 98.69% for CD73, 88.45% for CD90, and 96.30% for CD105. The protein level of secretome mesenchymal stem -like cells under hypoxia was measured at 5.56 μg/mL and 4.28 μg/mL for non-hypoxia. The liquid chromatography-mass spectrometry analysis identified 130 and 59 peptides from hypoxia and non-hypoxia of the human breastmilk stem cell secretome sequentially. Some important proteomics structures were found in the hypoxic human breastmilk stem cell secretome, such as transforming growth factor-β, VE-cadherin, and caspase.ConclusionThe human breastmilk cells contain mesenchymal stem-like cells and a high concentration of CD44, CD73, CD90, and CD105 as surface markers at third passage culture. The hypoxic hBSC secretome produces a higher protein level compare to non-hypoxia. The transforming growth factor -β was found in the hypoxic hBSC secretome as a modulator of VEGF-mediated angiogenesis.     

Modelling the binding mode of macrocycles: Docking and conformational sampling

Drug discovery is increasingly tackling challenging protein binding sites regarding molecular recognition and druggability, including shallow and solvent-exposed protein-protein interaction interfaces. Macrocycles are emerging as promising chemotypes to modulate such sites. Despite their chemical complexity, macrocycles comprise important drugs and offer advantages compared to non-cyclic analogs, hence the recent impetus in the medicinal chemistry of macrocycles. Elaboration of macrocycles, or constituent fragments, can strongly benefit from knowledge of their binding mode to a target. When such information from X-ray crystallography is elusive, computational docking can provide working models. However, few studies have explored docking protocols for macrocycles, since conventional docking methods struggle with the conformational complexity of macrocycles, and also potentially with the shallower topology of their binding sites. Indeed, macrocycle binding mode prediction with the mainstream docking software GOLD has hardly been explored. Here, we present an in-depth study of macrocycle docking with GOLD and the ChemPLP scores. First, we summarize the thorough curation of a test set of 41 protein-macrocycle X-ray structures, raising the issue of lattice contacts with such systems. Rigid docking of the known bioactive conformers was successful (three top ranked poses) for 92.7% of the systems, in absence of crystallographic waters. Thus, without conformational search issues, scoring performed well. However, docking success dropped to 29.3% with the GOLD built-in conformational search. Yet, the success rate doubled to 58.5% when GOLD was supplied with extensive conformer ensembles docked rigidly. The reasons for failure, sampling or scoring, were analyzed, exemplified with particular cases. Overall, binding mode prediction of macrocycles remains challenging, but can be much improved with tailored protocols. The analysis of the interplay between conformational sampling and docking will be relevant to the prospective modelling of macrocycles in general.     

Whole exome sequencing,in silico and functional studies confirm the association of the GJB2 mutation p.Cys169Tyr with deafness and suggest a role for the TMEM59 gene in the hearing process

The development of next generation sequencing techniques has facilitated the detection of mutations at an unprecedented rate. These efficient tools have been particularly beneficial for extremely heterogeneous disorders such as autosomal recessive non-syndromic hearing loss, the most common form of genetic deafness. GJB2 mutations are the most common cause of hereditary hearing loss. Amongst them the NM_004004.5: c.506G > A (p.Cys169Tyr) mutation has been associated with varying severity of hearing loss with unclear segregation patterns. In this study, we report a large consanguineous Emirati family with severe to profound hearing loss fully segregating the GJB2 missense mutation p.Cys169Tyr. Whole exome sequencing (WES), in silico, splicing and expression analyses ruled out the implication of any other variants and confirmed the implication of the p.Cys169Tyr mutation in this deafness family. We also show preliminary murine expression analysis that suggests a link between the TMEM59 gene and the hearing process. The present study improves our understanding of the molecular pathogenesis of hearing loss. It also emphasizes the significance of combining next generation sequencing approaches and segregation analyses especially in the diagnosis of disorders characterized by complex genetic heterogeneity.     

Efficient computation of subset selection probabilities with application to Cox regression

An efficient method is presented to compute the probabilityof selection of a specified subset from the set of all subsetsof a fixed size where the subsets are taken from a populationwhose units have varying individual probabilities of selection.The problem is motivated by the computation of the exact marginallikelihood for the Cox proportional hazards model.     

Proteomics-based synapse characterization: From proteins to circuits

The highly heterogeneous nature of neuronal cell types and their connections presents a major challenge to the characterization of neural circuits at the protein level. New approaches now enable an increasingly sophisticated dissection of cell type- and cellular compartment-specific proteomes, as well as the profiling of the protein composition of specific synaptic connections. Here, we provide an overview of these approaches and discuss how they hold considerable promise toward unravelling the molecular mechanisms of neural circuit formation and function. Finally, we provide an outlook of technological developments that may bring the characterization of synaptic proteomes at the single-synapse level within reach.     

Progress toward automated methyl assignments for methyl-TROSY applications

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BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences

'BLAST 2 Sequences', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 Sequences' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.++ +html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.