An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis

An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 1

Conceptualising production, productivity and technology in pharmacy practice: a novel framework for policy, education and research.

CONTEXT AND BACKGROUND: People and health systems worldwide face serious challenges due to shifting disease demographics, rising population demands and weaknesses in healthcare provision, including capacity shortages and lack of impact of healthcare services. These multiple challenges, linked with the global push to achieve universal health coverage, have made apparent the importance of investing in workforce development to improve population health and economic well-being. In relation to medicines, health systems face challenges in terms of access to needed medicines, optimising medicines use and reducing risk. In 2017, the International Pharmaceutical Federation (FIP) published global policy on workforce development ('the Nanjing Statements') that describe an envisioned future for professional education and training. The documents make clear that expanding the pharmacy workforce benefits patients, and continually improving education and training produces better clinical outcomes. AIMS AND PURPOSE: The opportunities for harnessing new technologies in pharmacy practice have been relatively ignored. This paper presents a conceptual framework for analysing production methods, productivity and technology in pharmacy practice that differentiates between dispensing and pharmaceutical care services. We outline a framework that may be employed to study the relationship between pharmacy practice and productivity, shaped by educational and technological inputs. METHOD AND RESULTS: The analysis is performed from the point of view of health systems economics. In relation to pharmaceutical care (patient-oriented practice), pharmacists are service providers; however, their primary purpose is not to deliver consultations, but to maximise the quantum of health gain they secure. Our analysis demonstrates that 'technology shock' is clearly beneficial compared with orthodox notions of productivity or incremental gain implementations. Additionally, the whole process of providing professional services using 'pharmaceutical care technologies' is governed by local institutional frames, suggesting that activities may be structured differently in different places and countries. DISCUSSION AND CONCLUSION: Addressing problems with medication use with the development of a pharmaceutical workforce that is sufficient in quantity and competence is a long-term issue. As a result of this analysis, there emerges a challenge about the profession's relationship with existing and emerging technical innovations. Our novel framework is designed to facilitate policy, education and research by providing an analytical approach to service delivery. By using this approach, the profession could develop examples of good practice in both developed and developing countries worldwide

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes. </p

Integrated nuclear proteomics and transcriptomics identifies S100A4 as a therapeutic target in acute myeloid leukemia

Inappropriate localization of proteins can interfere with normal cellular function and drive tumor development. To understand how this contributes to the development of acute myeloid leukemia (AML), we compared the nuclear proteome and transcriptome of AML blasts with normal human CD34+ cells. Analysis of the proteome identified networks and processes that significantly affected transcription regulation including misexpression of 11 transcription factors with seven proteins not previously implicated in AML. Transcriptome analysis identified changes in 40 transcription factors but none of these were predictive of changes at the protein level. The highest differentially expressed protein in AML nuclei compared with normal CD34+ nuclei (not previously implicated in AML) was S100A4. In an extended cohort, we found that over-expression of nuclear S100A4 was highly prevalent in AML (83%; 20/24 AML patients). Knock down of S100A4 in AML cell lines strongly impacted their survival whilst normal hemopoietic stem progenitor cells were unaffected. These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression or regulation of transcription that would not have been seen at the mRNA level. These data also suggest that S100A4 is essential for AML survival and could be a therapeutic target in AML

Broadening the horizon – level 2.5 of the HUPO-PSI format for molecular interactions

BACKGROUND: Molecular interaction Information is a key resource in modern biomedical research. Publicly available data have previously been provided in a broad array of diverse formats, making access to this very difficult. The publication and wide implementation of the Human Proteome Organisation Proteomics Standards Initiative Molecular Interactions (HUPO PSI-MI) format in 2004 was a major step towards the establishment of a single, unified format by which molecular interactions should be presented, but focused purely on protein-protein interactions. RESULTS: The HUPO-PSI has further developed the PSI-MI XML schema to enable the description of interactions between a wider range of molecular types, for example nucleic acids, chemical entities, and molecular complexes. Extensive details about each supported molecular interaction can now be captured, including the biological role of each molecule within that interaction, detailed description of interacting domains, and the kinetic parameters of the interaction. The format is supported by data management and analysis tools and has been adopted by major interaction data providers. Additionally, a simpler, tab-delimited format MITAB2.5 has been developed for the benefit of users who require only minimal information in an easy to access configuration. CONCLUSION: The PSI-MI XML2.5 and MITAB2.5 formats have been jointly developed by interaction data producers and providers from both the academic and commercial sector, and are already widely implemented and well supported by an active development community. PSI-MI XML2.5 enables the description of highly detailed molecular interaction data and facilitates data exchange between databases and users without loss of information. MITAB2.5 is a simpler format appropriate for fast Perl parsing or loading into Microsoft Excel

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify \u3e4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories