Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αßγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δßγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

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).

Nuclear factor of activated T-cell isoform expression and regulation in human myometrium.

BACKGROUND: During pregnancy, myometrial gene and protein expression is tightly regulated to accommodate fetal growth, promote quiescence and ultimately prepare for the onset of labour. It is proposed that changes in calcium signalling, may contribute to regulating gene expression and that nuclear factor of activated T-cell (NFAT) transcription factors (isoforms c1-c4) may be involved. Currently, there is little information regarding NFAT expression and regulation in myometrium. METHODS: This study examined NFAT isoform mRNA expression in human myometrial tissue and cells from pregnant women using quantitative PCR. The effects of the Ca(2+) ionophore A23187 and in vitro stretch (25 % elongation, static strain; Flexercell FX-4000 Tension System) on NFAT expression were determined in cultured human myometrial cells. RESULTS: Human myometrial tissue and cultured cells expressed NFATc1-c4 mRNA. NFATc2 gene expression in cultured cells was increased in response to 6 h stretch (11.5 fold, P < 0.001, n = 6) and calcium ionophore (A23187, 5 µM) treatment (20.6 fold, P < 0.001, n = 6). This response to stretch was significantly reduced (90 %, P < 0.001, n = 10) in the presence of an intracellular calcium chelator, BAPTA-AM (20 µM). CONCLUSIONS: These data suggest that NFATc2 expression is regulated by intracellular calcium and in vitro stretch, and that the stretch response in human myometrial cells is dependent upon intracellular calcium signalling pathways. Our findings indicate a potentially unique role for NFATc2 in mediating stretch-induced gene expression per se and warrant further exploration in relation to the mechanisms promoting uterine smooth muscle growth in early pregnancy and/or labour.

Human uterine and placental arteries exhibit tissue-specific acute responses to 17ß-estradiol and estrogen-receptor-specific agonists.

The discrete regulation of vascular tone in the human uterine and placental circulations is a key determinant of appropriate uteroplacental blood perfusion and pregnancy success. Humoral factors such as estrogen, which increases in the placenta and maternal circulation throughout human pregnancy, may regulate these vascular beds as studies of animal arteries have shown that 17ß-estradiol, or agonists of estrogen receptors (ER), can exert acute vasodilatory actions. The aim of this study was to compare how acute exposure to ER-specific agonists, and 17ß-estradiol, altered human placental and uterine arterial tone in vitro. Uterine and placental arteries were isolated from biopsies obtained from women with uncomplicated pregnancy delivering a singleton infant at term. Vessels were mounted on a wire myograph, exposed to the thromboxane receptor agonist U46619 (10(-6) M), and then incubated with incremental doses (5 min, 0.03-30 µM) of either 17ß-estradiol or agonists specific for the ERs ERα (PPT), ERß (DPN) or the G-protein-coupled estrogen receptor GPER-1 (G1). ERα and ERß mRNA expression was assessed. 17ß-estradiol, PPT and DPN each relaxed myometrial arteries (P < 0.05) in a manner that was partly endothelium-dependent. In contrast, 17ß-estradiol or DPN relaxed placental arteries (maximum relaxation to 42 ± 1.1 or 47.6 ± 6.53% of preconstriction, respectively) to a lesser extent than myometrial arteries (to 0.03 ± 0.03 or 8.0 ± 1.0%) and in an endothelial-independent manner whereas PPT was without effect. G1 exposure did not inhibit the constriction of myometrial nor placenta arteries. mRNA expression of ERα and ERß was greater in myometrial arteries than placental arteries. ER-specific agonists, and 17ß-estradiol, differentially modulate the tone of uterine versus placental arteries highlighting that estrogen may regulate human uteroplacental blood flow in a tissue-specific manner.

Age-related changes in the contractile and passive arterial properties of murine mesenteric small arteries are altered by caveolin-1 knockout.

Caveolin-1, an integral protein of caveolae, is associated with multiple cardiovascular signalling pathways. Caveolin-1 knockout (KO) mice have a reduced lifespan. As changes in artery structure and function are associated with ageing we have investigated the role of caveolin-1 ablation on age-related changes of small artery contractility and passive mechanical properties. Mesenteric small arteries isolated from 3 and 12-month wild-type (WT) and caveolin-1 KO mice were mounted on a pressure myograph and changes in passive and functional arterial properties were continuously monitored. In WT mice ageing was associated with a reduction in arterial contractility to noradrenaline which was reversed by inhibition of nitric oxide synthase with L-NNA. Similarly, in 3-month-old mice, caveolin-1 KO reduced contractility to noradrenaline by an L-NNA-sensitive mechanism. However, ageing in caveolin-1 KO mice was not associated with any further change in contractility. In WT mice ageing was associated with an increased passive arterial diameter and cross-sectional area (CSA), consistent with outward remodelling of the arterial wall, and a reduced arterial distensibility. Caveolin-1 ablation at 3 months of age resulted in similar changes in passive arterial properties to those observed with ageing in WT animals. However, ageing in caveolin-1 KO mice resulted in a reduced arterial CSA indicating different effects on passive structural characteristics from that observed in WT mice. Thus, caveolin-1 mice show abnormalities of small mesenteric artery function and passive mechanical characteristics indicative of premature vascular ageing. Moreover, caveolin-1 ablation modulates the age-related changes usually observed in mesenteric arteries of WT mice.

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.

Molecular pathways regulating contractility in rat uterus through late gestation and parturition.

OBJECTIVE: Endogenous uterine agonists can activate numerous signaling pathways to effect increased force. Our objective was to assess expression of key constituents of these pathways, in alliance with contractile function, through late gestation and during term and preterm labor. STUDY DESIGN: Using myography, we measured the response to 3 agonists compared with depolarization alone (K(+), 124 mEq/L) and calculated agonist/depolarization ratio. We measured gene expression using quantitative reverse transcription-polymerase chain reaction. RESULTS: Contractile responsiveness to depolarization alone, oxytocin, or endothelin-1 increased during pregnancy compared with nonpregnant animals. The agonist/depolarization ratio did not change during uterine activation or parturition. Inhibition of rhoA-associated kinase decreased responses to oxytocin in all tissues, but significantly more during uterine activation. Expression of rhoA and rhoA-associated kinase was increased significantly in active labor at term or preterm. CONCLUSION: The rhoA/rhoA-associated kinase pathway is a key regulator of uterine activation during labor and may be a useful target for the prevention of spontaneous preterm birth.

Regional identification of information flow termination of electrohysterographic signals: Towards understanding human uterine electrical propagation.

BACKGROUND AND OBJECTIVE: The uterine electrohysterogram (EHG) contains important information about electrical signal propagation which may be useful to monitor and predict the progress of pregnancy towards parturition. Directed information processing has the potential to be of use in studying EHG recordings. However, so far, there is no directed information-based estimation scheme that has been applied to investigating the propagation of human EHG recordings. To realize this, the approach of directed information and its reliability and adaptability should be scientifically studied. METHODS: We demonstrated an estimation scheme of directed information to identify the spatiotemporal relationship between the recording channels of EHG signal and assess the algorithm reliability initially using simulated data. Further, a regional identification of information flow termination (RIIFT) approach was developed and applied for the first time to extant multichannel EHG signals to reveal the terminal zone of propagation of the electrical activity associated with uterine contraction. RIIFT operates by estimating the pairwise directed information between neighboring EHG channels and identifying the location where there is the strongest inward flow of information. The method was then applied to publicly-available experimental data obtained from pregnant women with the use of electrodes arranged in a 4-by-4 grid. RESULTS: Our results are consistent with the suggestions from the previous studies with the added identification of preferential sites of excitation termination - within the estimated area, the direction of surface action potential propagation towards the medial axis of uterus during contraction was discovered for 72.15% of the total cases, demonstrating that our RIIFT method is a potential tool to investigate EHG propagation for advancing our understanding human uterine excitability. CONCLUSIONS: We developed a new approach and applied it to multichannel human EHG recordings to investigate the electrical signal propagation involved in uterine contraction. This provides an important platform for future studies to fill knowledge gaps in the spatiotemporal patterns of electrical excitation of the human uterus.

Strategies for Peptide-Mediated Cargo Delivery to Human Smooth Muscle Cells.

Diseases involving dysfunction of smooth muscle cells present a major health and socioeconomic burden, and have remained stubbornly resistant to standard therapeutic strategies. Examples include many cardiovascular diseases and spontaneous preterm birth, a complication affecting up to 11% of all pregnancies worldwide. This fuels the continued search for new drug delivery strategies to treat these conditions. The use of cell penetrating peptides (CPPs) for this purpose remains a promising, if as yet unrealized, avenue to explore. In part, this may relate to a paucity of studies investigating the application of CPPs as drug delivery vectors to human smooth muscle cells and tissues. We have sought to address this knowledge gap by reporting methods for examining the uptake of different CPP-cargo vectors to human uterine and vascular smooth muscle cells. In particular, we report here (a) that four different CPP-fluorophore conjugates, spanning masses of 1309-3435 Da, and net charges of +2 to +7, can be delivered to human isolated uterine smooth muscle cells without inducing cell toxicity; (b) that the cargo delivered by such CPPs can be fluorescent moieties and/or biologically active peptides; (c) that CPP delivery in a short time frame to native smooth muscle cells in human tissues ex vivo can be achieved. Further exploration of CPPs as tools to facilitate targeted drug delivery to native human smooth muscle tissues will assist in improving our understanding of scientific mechanisms underlying major diseases involving smooth muscle dysfunction as well as facilitating therapeutic investigations.

Possible dual roles for prostacyclin in human pregnancy and labor.

During pregnancy, the muscular layer of the uterine wall known as the myometrium, which is composed mainly of smooth muscle cells, is maintained in a state of relative quiescence. A switch from myometrial quiescence to myometrial activation is required to establish uterine contractions during labor. Researchers have long been perplexed by the fact that the major prostaglandin produced by the uterus just prior to labor, prostacyclin, is a smooth muscle relaxant. In this issue of the JCI, Fetalvero et al. provide data that they propose explains this paradox, at least in part (see the related article beginning on page 3966). The authors examined uterine tissue from pregnant women near term and found that prostacyclin stimulation, which raises cAMP levels that were previously thought to affect only myometrial quiescence, can promote myometrial activation over time by increasing the expression of a select group of proteins thought to be indicative of a uterine contractile state.

Three-dimensional electron microscopic reconstruction of intracellular organellar arrangements in vascular smooth muscle--further evidence of nanospaces and contacts.

The sarcoplasmic reticulum (SR) of smooth muscle is crucial for appropriate regulation of Ca(2+) signalling. In visceral and vascular smooth muscles the SR is known to periodically lie in close register, within a few nanometres, to the plasma membrane. Recent work has focussed on reconstructions of the ultrastructural arrangement of this so-called peripheral SR that may be important for the genesis of phenomena such as Ca(2+) sparks. Here, we turn our attention to vascular smooth muscle and explore the 3-dimensional (3D) ultrastructural positioning of SR found deeper in the cell that is involved in the propagation of Ca(2+) waves. We use digital reconstruction and volume rendering of serial electron microscopic sections from isolated resistance arteries, pressurized in vitro to mimic cellular geometric conformations anticipated in vivo, to map SR positioning. We confirm that these central portions of SR are in close register with mitochondria and the nucleus with all three organelles tightly enveloped by a myofilament/cytoskeletal lattice. Nanospacings between the SR and individual mitochondria are visible and in three dimensions as the SR contorts to accommodate these organelles. Direct connection of the SR and nuclear membranes is confirmed. Such 3D positioning of centrally located SR further informs us of its likely role in the manifestation of spatiotemporal Ca(2+) dynamics: signal encoding may be facilitated by spatially directed release of Ca(2+) to influence several processes crucial to vascular smooth muscle and resistance artery function including myofilament activation by Ca(2+) waves, mitochondrial respiration and gene transcription.

Are animal models relevant to key aspects of human parturition?

Preterm birth remains the most serious complication of pregnancy and is associated with increased rates of infant death or permanent neurodevelopmental disability. Our understanding of the regulation of parturition remains inadequate. The scientific literature, largely derived from rodent animal models, suggests two major mechanisms regulating the timing of parturition: the withdrawal of the steroid hormone progesterone and a proinflammatory response by the immune system. However, available evidence strongly suggests that parturition in the human has significantly different regulators and mediators from those in most of the animal models. Our objectives are to critically review the data and concepts that have arisen from use of animal models for parturition and to rationalize the use of a new model. Many animal models have contributed to advances in our understanding of the regulation of parturition. However, we suggest that those animals dependent on progesterone withdrawal to initiate parturition clearly have a limitation to their translation to the human. In such models, a linear sequence of events (e.g., luteolysis, progesterone withdrawal, uterine activation, parturition) gives rise to the concept of a "trigger" mechanism. Conversely, we propose that human parturition may arise from the concomitant maturation of several systems in parallel. We have termed this novel concept "modular accumulation of physiological systems" (MAPS). We also emphasize the urgency to determine the precise role of the immune system in the process of parturition in situations other than intrauterine infection. Finally, we accentuate the need to develop a nonprimate animal model whose physiology is more relevant to human parturition. We suggest that the guinea pig displays several key physiological characteristics of gestation that more closely resemble human pregnancy than do currently favored animal models. We conclude that the application of novel concepts and new models are required to advance translational research in parturition.

Uterine artery function in a mouse model of pregnancy complicated by diabetes.

It has been demonstrated previously that endothelium-dependent vasodilatation is impaired in myometrial arteries from women with gestational diabetes, which may play a role in mediating complications observed in diabetic pregnancies. It is not known which aspects of endothelium-dependent vasodilatation are impaired, thus a mouse model of pregnancy complicated by streptozotocin-induced diabetes was established to investigate underlying mechanisms. Uterine arteries from term-pregnant, diabetic and control C57Bl6/J mice were assessed using acetylcholine (ACh; 10(-10)-10(-5)M) in the presence or absence of a nitric oxide (NO) synthase inhibitor (L-NNA; 10(-5)M), a cyclooxygenase (COX) inhibitor (indomethacin; 10(-5)M) or the two in combination. Sensitivity to ACh was comparable between diabetic and control mice. However, the contribution of endothelium-dependent vasodilators was significantly altered. L-NNA significantly inhibited the relaxation of arteries from diabetic compared to control mice (65+/-11% vs 18+/-6%; p<.05). L-NNA and indomethacin significantly inhibited the relaxation of arteries from diabetic mice compared to control (87+/-5% vs 33+/-14%; p<0.05). These data indicate that endothelium-dependent relaxation of the uterine artery of control, pregnant mice was largely mediated by the non-NO/non-COX component. Surprisingly, arteries from diabetic mice were primarily dependent on NO, which may affect compensatory capacity as the disease progresses.

Relationship between gene expression and function of uterotonic systems in the rat during gestation, uterine activation and both term and preterm labour.

We have documented gestation- and labour- (preterm and term) dependent changes in expression of genes encoding contraction associated proteins in the rat uterus and correlated these changes with various parameters of uterine contractility. The data demonstrate increased expression of contractile agonist systems concurrent with decreased expression of relaxant systems after gestational day 20. Significant increases in expression of oxytocin (OT), its receptor (OTR), prostaglandin (PG) H synthase isoform 1 (PGHS-1) and PGF(2alpha) receptor (FP) occurred first, followed by increases in PGHS-2, connexin-43, endothelin-1 (ET-1) and the ET-1 receptor isoform ET(A). Expression of OTR and FP was significantly reduced during mid-gestation compared to non-pregnant animals. Expression of inducible nitric oxide synthase (iNOS) increased significantly during pregnancy then decreased concurrently with the increase in OTR and FP. Functional changes in uterine contractility accompany changes in gene expression. OT was the most potent contractile stimulant. Sensitivity of uterine strips to OT was reduced in early and mid-pregnancy then increased at uterine activation. Progesterone antagonist-induced preterm labour caused changes similar to those at normal term. Comparison of mRNA transcripts in separated endometrium and myometrium suggested that the endometrium is an important regulator of myometrial contractility, analogous to the relationship between endothelium and vascular smooth muscle. This novel combination of functional and genetic expression analyses provides new insight into the physiology of parturition.

Modulation of caveolin-1 expression can affect signalling through the phosphatidylinositol 3-kinase/Akt pathway and cellular proliferation in response to insulin-like growth factor I.

The IGFs mediate their effects on cell function through the type I IGF receptor and numerous intracellular signalling molecules, including the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. The type I IGF receptor also binds to the caveolae protein caveolin-1, but the impact of caveolae on IGF/PI-3K/Akt signalling remains controversial. We have examined the effect of complete (knockout) and partial (knockdown) caveolin-1 deficiency on cellular IGF effects mediated via the PI-3K/Akt pathway. Under basal conditions, caveolin-1-deficient mouse embryonic fibroblast cells [MF(-/-)] incorporated significantly more [3H]thymidine than wild-type mouse embryonic fibroblast cells [MF(+/+)]; however, small hairpin RNA-mediated knockdown of caveolin-1 (80% reduction) in 3T3L1 fibroblasts had no effect on basal proliferation. Interestingly, IGF-I induced proliferation was similar in MF(-/-) and MF(+/+) cells, whereas caveolin-1 knockdown promoted a hyperproliferative response to IGF-I [pkDCav3T3L1(80) 12.4+/-0.4-fold; pkDShuffle3T3L1 4.3+/-0.2-fold induction; P<0.01]. Immunoblot analysis showed that caveolin-1 knockdown had no affect on Akt expression or activation. However, in MF(-/-) cells, IGF-I-stimulated phosphorylation of Akt was reduced despite up-regulated Akt levels. Further investigation demonstrated that caveolin knockout up-regulated Akt-2 and Akt-3 isoform expression, but Akt-1 expression was down-regulated; interestingly, coimmunoprecipitation studies revealed Akt-1 as the predominant isoform to be phosphorylated in response to IGF-I. In summary, caveolin-1 deficiency promotes a hyperproliferative response to IGF-I that is unrelated to Akt expression/activation. However, cells that lack caveolin are able to respond appropriately to IGF-I through compensatory changes in Akt isoform expression. These data posit caveolin-1 as a component of the IGF/PI-3K/Akt signalling modulus regulating cellular proliferation with implications for diseases, including cancers, which have altered caveolin expression.

Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB.

Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1ß-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.

Depletion of Myofibril-Associated Proteins Using Selective Protein Extraction as a Tool in Cardiac Proteomics.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.