The Generation of a Comprehensive Spectral Library for the Analysis of the Guinea Pig Proteome by SWATH-MS.

Advances in liquid chromatography-mass spectrometry have facilitated the incorporation of proteomic studies to many biology experimental workflows. Data-independent acquisition platforms, such as sequential window acquisition of all theoretical mass spectra (SWATH-MS), offer several advantages for label-free quantitative assessment of complex proteomes over data-dependent acquisition (DDA) approaches. However, SWATH data interpretation requires spectral libraries as a detailed reference resource. The guinea pig (Cavia porcellus) is an excellent experimental model for translation to many aspects of human physiology and disease, yet there is limited experimental information regarding its proteome. To overcome this knowledge gap, a comprehensive spectral library of the guinea pig proteome is generated. Homogenates and tryptic digests are prepared from 16 tissues and subjected to >200 DDA runs. Analysis of >250 000 peptide-spectrum matches resulted in a library of 73 594 peptides from 7666 proteins. Library validation is provided by i) analyzing externally derived SWATH files (https://doi.org/10.1016/j.jprot.2018.03.023) and comparing peptide intensity quantifications; ii) merging of externally derived data to the base library. This furnishes the research community with a comprehensive proteomic resource that will facilitate future molecular-phenotypic studies using (re-engaging) the guinea pig as an experimental model of relevance to human biology. The spectral library and raw data are freely accessible in the MassIVE repository (MSV000083199).

Preparing pharmacists for the Community Pharmacist Consultation Service: a questionnaire survey.

OBJECTIVES: The Community Pharmacist Consultation Service launched in England in 2019. Patients requiring urgent care were referred from National Health Service-based telephone/digital triage or general practice to a community pharmacist, who provided a consultation, which could include a physical examination. The aim of the study was to evaluate the effectiveness of a learning programme to prepare community pharmacists for the service. METHODS: Learning programme participants were invited to complete an online survey shortly after the workshop and another survey 3 months later. The survey collected opinions on aspects of the programme, including Likert-type statements and free text questions. The 3-month follow-up survey explored how the programme had helped pharmacists change their practice. Data were analysed in SPSS (v.25; IBM) with inferential statistics used to compare subgroups. Open comments were analysed qualitatively. KEY FINDINGS: The learning programme addressed participants' learning needs including history-taking, clinically observing the patient, performing physical examinations, structuring a consultation, safety-netting, and documenting consultations. Barriers to using skills acquired included low service uptake and a lack of equipment to perform physical examinations. While many participants recognised the importance of skills to provide person-centred care, some participants did not appear to recognise the shift in policy to a more clinical role. CONCLUSIONS: The learning programme resulted in increased confidence and a recognition of a shift in the policy vision for community pharmacist roles. Although some pharmacists appeared to embrace this, others have yet to fully appreciate the need to adapt to be ready for the opportunities that this service can provide.

Acetylation of heat shock protein 20 (Hsp20) regulates human myometrial activity.

Phosphorylation of heat shock protein 20 (Hsp20) by protein kinase A (PKA) is now recognized as an important regulatory mechanism modulating contractile activity in the human myometrium. Thus agonists that stimulate cyclic AMP production may cause relaxation with resultant beneficial effects on pathologies that affect this tissue such as the onset of premature contractions prior to term. Here we describe for the first time that acetylation of Hsp20 is also a potent post-translational modification that can affect human myometrial activity. We show that histone deacetylase 8 (HDAC8) is a non-nuclear lysine deacetylase (KDAC) that can interact with Hsp20 to affect its acetylation. Importantly, use of a selective linkerless hydroxamic acid HDAC8 inhibitor increases Hsp20 acetylation with no elevation of nuclear-resident histone acetylation nor marked global gene expression changes. These effects are associated with significant inhibition of spontaneous and oxytocin-augmented contractions of ex vivo human myometrial tissue strips. A potential molecular mechanism by which Hsp20 acetylation can affect myometrial activity by liberating cofilin is described and further high-lights the use of specific effectors of KDACs as therapeutic agents in regulating contractility in this smooth muscle.

Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells.

Recently we reported that the expression of the protein kinase A (PKA) regulatory subunit RIIα is dynamically regulated in human smooth muscle cells of the uterus. We showed that expression levels of mRNA/protein were substantially increased during pregnancy and decreased upon labour, changes that were mirrored by particulate type II PKA activity. This implied an important role for RIIα in maintaining uterine quiescence during pregnancy. Consequently the purpose of the present study was to identify potential mechanisms by which expression of the RIIα gene was regulated in this tissue. We indicate here that the three SpI-III (GC) binding domains within the proximal promoter region of the human RIIα gene may play important roles in modulating expression of the gene in human myometrial cells. We show that all three GC binding domains are involved in binding Sp1, Sp3, histone deacetylase (HDACs) 1/2 and RbAp48 transcriptional complexes. The functional significance of these binding domains was further analysed employing in vitro luciferase reporter assays with full-length/truncated RIIα promoter constructs. Importantly we show that treatment of primary human myometrial cell cultures with the general class I/II HDAC inhibitor trichostatin A results in an increase in mRNA/protein levels. Moreover the increase in mRNA levels appeared to be preceded by an increase in aH3, PolIIa, Sp3 and HDAC 2 binding to the three SpI-III (GC) binding sites within the RIIα promoter. These results enable us to provide a model whereby RIIα expression is epigenetically regulated in human myometrial smooth muscle cells by histone deacetylase(s) activity within the GC-rich proximal promoter region of the gene.

IGF signalling and endocytosis in the human villous placenta in early pregnancy as revealed by comparing quantum dot conjugates with a soluble ligand.

A complex combination of trafficking and signalling occurs at the surface of the placenta. The system delivers maternal nutrients to the fetus and facilitates gaseous exchange, whilst mediating signal transduction to support and stimulate the growth of the placenta itself. IGF-I is acknowledged as a maternally-derived ligand important in the regulation of placental growth. Here we show that quantum dots bearing IGF can stimulate IGF receptor (IGF1R) phosphorylation in the syncytio- (maternal-facing) and cyto- (fetal-facing) trophoblast bilayer that forms the outer boundary of the placenta, in a distribution similar to the one resulting from exposure to a soluble ligand. The conjugates are internalised by a clathrin-dependent pathway and delivered to a syncytioplasmic compartment that differs from conventional late endosomes and lysosomes. Two discrete downstream responses are evident in different cellular compartments: phosphorylation of P70S6K in the non-proliferative syncytiotrophoblast and of AKT in the cytotrophoblast. Co-conjugation of IGF-quantum dots with an RGD-containing ligand permits penetration beyond the syncytium, into the cytoplasm of the underlying cytotrophoblast. These data reveal the existence of a trans-syncytial pathway that allows maternal mitotic signals to penetrate to the inner progenitor cells, which must proliferate to support placental and consequently fetal growth.

Vasoactive intestinal peptide shapes first-trimester placenta trophoblast, vascular, and immune cell cooperation.

BACKGROUND AND PURPOSE: Extravillous trophoblast (EVT) cells are responsible for decidual stromal invasion, vascular transformation, and the recruitment and functional modulation of maternal leukocytes in the first-trimester pregnant uterus. An early disruption of EVT function leads to placental insufficiency underlying pregnancy complications such as preeclampsia and fetal growth restriction. Vasoactive intestinal peptide (VIP) is a vasodilating and immune modulatory factor synthesized by trophoblast cells. However, its role in first-trimester placenta has not been explored. Here, we tested the hypothesis that VIP is involved in first-trimester EVT outgrowth, spiral artery remodelling, balancing angiogenesis, and maintenance of immune homeostasis. EXPERIMENTAL APPROACH: First-trimester placental tissue (five to nine weeks of gestation) was collected, and was used for EVT outgrowth experiments, immunofluorescence, isolation of decidual natural killer (dNK) cells and decidual macrophages (dMA), and functional assays. Peripheral blood monocytes were differentiated with GM-CSF and used for angiogenesis assays. KEY RESULTS: In decidua basalis, VIP+ EVT were observed sprouting from cell columns and lining spiral arterioles. EVT migrating from placental explants were also VIP+. VIP increased EVT outgrowth and IL-10 release, whereas it decreased pro-inflammatory cytokine production in EVT, dNK cells, and dMA. VIP disrupted endothelial cell networks, both directly and indirectly via an effect on macrophages. CONCLUSION AND IMPLICATIONS: The results suggest that VIP assists the progress of EVT invasion and vessel remodelling in first-trimester placental bed in an immunologically "silent" milieu. The effects of VIP in the present ex vivo human placental model endorse its potential as a therapeutic candidate for deep placentation disorders.

A role for cytoskeletal protein acetylation in modulating myometrial activity.

Posttranslational modifications (PTMs) of proteins by phosphorylation are a well-established mechanism by which their activities can be regulated to affect cellular physiology. However, it is becoming increasingly evident that PTMs of proteins by acetylation of lysine residues is also a key effecter in regulating their functional abilities. The best characterized case of this is the epigenetic effects of histone acetyltransferases and deacetylases on gene expression via modulation of nuclear histone acetylation and chromatin remodeling. However, recent published evidence now strongly implicates an important role for nonhistone acetylation in regulating cellular function. In this review, we have considered the potential for regulating myometrial activity not only by epigenetic mechanisms but also by nonepigenetic protein acetylation processes that could directly affect the contractile machinery within these smooth muscle cells.

Lysine deacetylase inhibition promotes relaxation of arterial tone and C-terminal acetylation of HSPB6 (Hsp20) in vascular smooth muscle cells.

There is increasing interest in establishing the roles that lysine acetylation of non nuclear proteins may exert in modulating cell function. Lysine deacetylase 8 (KDAC8), for example, has been suggested to interact with α-actin and control the differentiation of smooth muscle cells. However, a direct role of smooth muscle non nuclear protein acetylation in regulating tone is unresolved. We sought to define the actions of two separate KDAC inhibitors on arterial tone and identify filament-interacting protein targets of acetylation and association with KDAC8. Compound 2 (a specific KDAC8 inhibitor) or Trichostatin A (TSA, a broad-spectrum KDAC inhibitor) inhibited rat arterial contractions induced by phenylephrine (PE) or high potassium solution. In contrast to the predominantly nuclear localization of KDAC1 and KDAC2, KDAC8 was positioned in extranuclear areas of native vascular smooth muscle cells. Several filament-associated proteins identified as putative acetylation targets colocalized with KDAC8 by immunoprecipitation (IP): cortactin, α-actin, tropomyosin, HSPB1 (Hsp27) and HSPB6 (Hsp20). Use of anti-acetylated lysine antibodies showed that KDAC inhibition increased acetylation of each protein. A custom-made antibody targeting the C-terminal acetylated lysine of human HSPB6 identified this as a novel target of acetylation that was increased by KDAC inhibition. HSPB6 phosphorylation, a known vasodilatory modification, was concomitantly increased. Interrogation of publicly available mass spectrometry data identified 50 other proteins with an acetylated C-terminal lysine. These novel data, in alliance with other recent studies, alert us to the importance of elucidating the mechanistic links between changes in myofilament-associated protein acetylation, in conjunction with other posttranslational modifications, and the regulation of arterial tone.

Expression of the GTP-binding protein Gαs in human myometrial cells is regulated by ubiquitination and protein degradation: involvement of proteasomal inhibition by trichostatin A.

In this study, we show that myometrial transcriptional complexes consisting of Sp1, Sp3, histone deacetylase (HDAC)1/2, RbAp48, and mSin3A are recruited to 4 out of the 6 Sp1-4 sites within the Gαs promoter. Moreover disruption in the binding of these complexes via mithramycin administration results in a substantial decrease in expression of Gαs proteins in myometrial cell cultures. In many instances, these transcriptional regulatory complexes repress expression of genes having a high CG content within their promoter region. This repression can be attenuated by inhibition of HDAC activity by the class I/II HDAC inhibitor trichostatin A (TSA) resulting in increased gene transcription. However, although a substantial increase in Gαs protein levels was observed upon administration of TSA to primary cultures of human myometrial cells, this was not preceded by an increase in messenger RNA (mRNA) and thus an elevation in gene transcription. Importantly the increase in Gαs protein levels occurred via ubiquitination and inhibition of proteasomal activity, indicating that this pathway is also involved in regulating Gαs protein expression during pregnancy and parturition.