Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation.

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.

A Quality Improvement Project to Reduce Antibiotic Exposure in Premature Neonates.

BACKGROUND AND OBJECTIVES: Overuse of antibiotics in NICUs is a problem worldwide. Unnecessary antibiotic exposure leads to resistance, changes in the microbiome, and increases the risk of bronchopulmonary dysplasia, retinopathy of prematurity, periventricular leukomalacia, necrotizing enterocolitis, late-onset sepsis (LOS), and mortality in neonates. We aimed to safely reduce the antibiotic usage rate (AUR) in our level IV unit by 10% by December 2018. METHODS: A multidisciplinary quality improvement project took place as part of a Vermont Oxford Network initiative in 2018. Multiple interventions took place, including identification of variations in practices and subsequent standardization through the creation of early onset and LOS guidelines, mass education, improved visibility of the guidelines, and standardized documentation. The main outcome measure for this project was the AUR for infants born <35 weeks' gestation expressed as antibiotic doses per 1000 patient days. RESULTS: The AUR decreased from a mean of 524 to 394, for a decrease of 24.8%. Results have been sustained for 3 years. Main contributors that led to the sustained success include decreasing the overall use of antibiotics for early onset sepsis, as well as the duration when cultures are negative. The number of LOS courses also decreased slightly. We noted no cases of inadequately treated sepsis resulting in subsequent positive cultures. CONCLUSIONS: Creation of guidelines with mass education and ongoing feedback/monitoring can result in a safe reduction of AUR in the NICU.

The fear of social stigma experienced by men: a barrier to male involvement in antenatal care in Misungwi District, rural Tanzania.

BACKGROUND: Evidence has shown that male involvement is associated with improved maternal health outcomes. In rural Tanzania, men are the main decision makers and may determine women's access to health services and ultimately their health outcomes. Despite efforts geared towards enhancing male participation in maternal health care, their involvement in antenatal care (ANC) remains low. One barrier that impacts men's participation is the fear and experience of social stigma. This study, builds on previous findings about men's perspectives in attending antenatal care appointments in Misungwi district in Tanzania, examining more closely the fear of social stigma amongst men attending ANC together with their partners. METHODS: Twelve individual interviews and five focus group discussions were conducted using semi-structured questionnaires with fathers and expectant fathers. In-depth interviews were conducted with health providers, volunteer community health workers and village leaders. Interviews were audiotaped, and transcripts were transcribed and translated to English. Transcripts were organized in NVivo V.12 then analyzed using thematic approach. RESULTS: Three main themes were found to create fear of social stigma for men: 1. Fear of HIV testing; 2. Traditional Gender Norms and 3. Insecurity about family social and economic status. CONCLUSION: Respondent's experiences reveal that fear of social stigma is a major barrier to attend ANC services with their partners. Attention must be given to the complex sociocultural norms and social context that underly this issue at the community level. Strategies to address fear of social stigma require an understanding of the real reasons some men do not attend ANC and require community engagement of community health workers (CHWs), government officials and other stakeholders who understand the local context.

Glucagon-like peptide-1 preserves coronary microvascular endothelial function after cardiac arrest and resuscitation: potential antioxidant effects.

Glucagon-like peptide-1 (GLP-1) has protective effects in the heart. We hypothesized that GLP-1 would mitigate coronary microvascular and left ventricular (LV) dysfunction if administered after cardiac arrest and resuscitation (CAR). Eighteen swine were subjected to ventricular fibrillation followed by resuscitation. Swine surviving to return of spontaneous circulation (ROSC) were randomized to receive an intravenous infusion of either human rGLP-1 (10 pmol·kg(-1)·min(-1); n = 8) or 0.9% saline (n = 8) for 4 h, beginning 1 min after ROSC. CAR caused a decline in coronary flow reserve (CFR) in control animals (pre-arrest, 1.86 ± 0.20; 1 h post-ROSC, 1.3 ± 0.05; 4 h post-ROSC, 1.25 ± 0.06; P < 0.05). GLP-1 preserved CFR for up to 4 h after ROSC (pre-arrest, 1.31 ± 0.17; 1 h post-ROSC, 1.5 ± 0.01; 4 h post-ROSC, 1.55 ± 0.22). Although there was a trend toward improvement in LV relaxation in the GLP-1-treated animals, overall LV function was not consistently different between groups. 8-iso-PGF(2α), a measure of reactive oxygen species load, was decreased in post-ROSC GLP-1-treated animals [placebo, control (NS): 38.1 ± 1.54 pg/ml; GLP-1: 26.59 ± 1.56 pg/ml; P < 0.05]. Infusion of GLP-1 after CAR preserved coronary microvascular and LV diastolic function. These effects may be mediated through a reduction in oxidative stress.

Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2.

BACKGROUND: Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. PRINCIPAL FINDINGS: The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. CONCLUSIONS/SIGNIFICANCE: S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.

A Solanum tuberosum inositol phosphate kinase (StITPK1) displaying inositol phosphate-inositol phosphate and inositol phosphate-ADP phosphotransferase activities.

We describe a multifunctional inositol polyphosphate kinase/phosphotransferase from Solanum tuberosum, StITPKalpha (GenBank accession number: EF362784), hereafter called StITPK1. StITPK1 displays inositol 3,4,5,6-tetrakisphosphate 1-kinase activity: K(m) = 27 microM, and V(max) = 19 nmol min(-1) mg(-1). The enzyme displays inositol 1,3,4,5,6-pentakisphosphate 1-phosphatase activity in the absence of a nucleotide acceptor and inositol 1,3,4,5,6-pentakisphosphate-ADP phosphotransferase activity in the presence of physiological concentrations of ADP. Additionally, StITPK1 shows inositol phosphate-inositol phosphate phosphotransferase activity. Homology modelling provides a structural rationale of the catalytic abilities of StITPK1. Inter-substrate transfer of phosphate groups between inositol phosphates is an evolutionarily conserved function of enzymes of this class.

Sustained hypertension increases the density of AMPA receptor subunit, GluR1, in baroreceptive regions of the nucleus tractus solitarii of the rat.

AMPA-type glutamate receptors in the nucleus tractus solitarii (NTS) are necessary for the baroreceptor reflex, a primary mechanism for homeostatic regulation of blood pressure. Within NTS, the GluR1 subunit of the AMPA receptor is found primarily in dendritic spines. We previously showed that both GluR1 and dendritic spine density are increased in NTS of spontaneously hypertensive rats (SHRs). We hypothesize that both receptor and synaptic plasticity are induced by a sustained elevation in arterial pressure. To test the general nature of this hypothesis, we examined whether similar changes in GluR1 density are found in a renovascular model of hypertension, the DOCA-salt rat, and if these changes are preventable by normalizing blood pressure with hydralazine, a peripherally acting vasodilator. Using immunoperoxidase detection, GluR1 appears as small puncta at the light microscopic level, and is found in dendritic spines at the ultrastructural level. Following the development of hypertension, GluR1 spine and puncta counts were significantly greater in DOCA-salt rats than controls. Hydralazine treatment (4-5 weeks) prevented the development of hypertension in DOCA-salt rats and reduced blood pressure of SHRs to normotensive levels. The density of GluR1 puncta in the NTS was significantly reduced by hydralazine treatment in the SHR model. These results show that hypertension alters dendritic spines containing AMPA-type glutamate receptors within NTS, suggesting that adjustments in GluR1 expression within NTS are part of the synaptic adaptations to the hypertensive state.

Kainate receptors are primarily postsynaptic to SP-containing axon terminals in the trigeminal dorsal horn.

Kainate receptors (KARs) are involved in the modulation and transmission of nociceptive information from peripheral afferents to neurons in the spinal cord and trigeminal dorsal horns. KARs are found at both pre- and postsynaptic sites in the dorsal horn. We hypothesized that KARs and Substance P (SP), a modulatory neuropeptide that is used as a marker of nociceptive afferents, have a complex interactive relationship. To determine the cellular relationship and connectivity between KARs and SP afferents, we used electron microscopic dual immunocytochemical analysis to examine the ultrastructural localization of KAR subunits GluR5, 6 and 7 (GluR5,6,7) in relation to SP within laminae I and II in the rat trigeminal dorsal horn. KARs were distributed both postsynaptically in dendrites and somata (51% of GluR5,6,7 immunoreactive (-ir) profiles) and presynaptically in axons and axon terminals (45%). We also found GluR5,6,7-ir glial profiles (5%). The majority of SP-ir profiles were presynaptic axons and axon terminals. SP-ir dendritic profiles were rare, yet 23% contained GluR5,6,7 immunoreactivity. GluR5,6,7 and SP were also colocalized at presynaptic sites (18% of GluR5,6,7-ir axons and axon terminals contained SP; while 11% of SP-ir axons and axon terminals contained GluR5,6,7). The most common interaction between KARs and SP we observed was GluR5,6,7-ir dendrites contacted by SP-ir axon terminals; 54% of the dendritic targets of SP-ir axon terminals were GluR5,6,7-ir. These results provide anatomical evidence that KARs primarily mediate nociceptive transmission postsynaptic to SP-containing afferents and may also modulate the presynaptic release of SP and glutamate in trigeminal dorsal horn.

Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein.

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.

Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes.

The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.

Most neurons in the nucleus tractus solitarii do not send collateral projections to multiple autonomic targets in the rat brain.

The nucleus tractus solitarii (NTS) receives primary visceral afferents and sends projections to other autonomic nuclei at all levels of the neuroaxis. However, it is unknown if distinct populations of NTS neurons project to individual autonomic targets or if individual neurons in the NTS project to multiple autonomic targets. Understanding the basic circuitry of visceral reflex pathways is essential for the analyses of functional central autonomic networks. We examined projections from the NTS to autonomic targets within the hypothalamus (paraventricular nucleus, PVN), pons (parabrachial nucleus, PB), and medulla (caudal ventrolateral medulla, CVL) using retrograde tracing and immunohistochemistry. Dual retrograde tracer microinjections were made into pairs of targets (PVN + CVL; PVN + PB; PB + CVL), and the pattern of retrograde labeling was examined within NTS. The extent of collateralization, seen as dual retrogradely labeled neurons, was negligible for combined PVN and CVL injections and increased for injections combining PB with either PVN or CVL, but the majority of NTS neurons project to only one autonomic target. Immunohistochemistry for tyrosine hydroxylase (TH) was used to examine the pattern of TH-immunoreactivity (TH-ir) within retrogradely labeled NTS neurons. TH-ir was seen predominantly in projections to PVN, to a lesser degree in projections to PB, and was largely absent from projections to CVL. The percentage of dual retrogradely labeled neurons displaying TH-ir corresponded to the target displaying the most TH-ir, and TH-ir was not predictive of collateralization. Together, these results indicate that NTS neurons project to individual autonomic targets in the brain.

Endomorphin-2 axon terminals contact mu-opioid receptor-containing dendrites in trigeminal dorsal horn.

The endomorphins represent a novel group of endogenous opioid peptides that have high affinity for the mu-opioid receptor (MOR1). Endomorphin-2 is present in high density in the spinal and trigeminal dorsal horns and is localized to primary afferents. If endomorphin-2 were an endogenous ligand for the MOR1, we would expect to find the receptor at cellular sites in close association with the peptide. We used dual-labeling immunocytochemical methods combined with electron microscopy to determine if a cellular substrate exists for functional interactions between endomorphin-2 and MOR1. We confirmed the localization of endomorphin-2 to unmyelinated axons and axon terminals in the trigeminal dorsal horn. A small proportion of these endomorphin-2 axons contained MOR1, but many of the dendritic targets of endomorphin-2 terminals contained MOR1. Consistent with previous studies, endomorphin-2 was contained primarily in dense core vesicles and MOR1 was located primarily at non-synaptic sites. These morphological characteristics are consistent with the hypothesis that peptides are released extra-synaptically and their receptors may be located at sites distal to the synaptic junction. These anatomical data support the hypothesis that endomorphin-2 is a ligand for MORs in the trigeminal dorsal horn, particularly at postsynaptic sites.

Structural changes in AMPA-receptive neurons in the nucleus of the solitary tract of spontaneously hypertensive rats.

The baroreceptor reflex is critical for homeostatic regulation of blood pressure and is initiated centrally by glutamate release from baroreceptive afferents onto neurons in the nucleus of the solitary tract that activates AMPA-type glutamate receptors. The GluR1 subunit of the AMPA receptor is located at postsynaptic sites within the nucleus of the solitary tract, particularly in dendritic spines, which are important sites for synaptic plasticity. We tested whether the distribution of GluR1 changes after sustained hypertension, which alters baroreceptor afferent activity. We examined the distribution of GluR1 in the nucleus of the solitary tract of both hypertensive (spontaneously hypertensive) and normotensive (Wistar-Kyoto) rats at the light microscopic and electron microscopic levels. There were more GluR1-containing dendritic spines in the nucleus of the solitary tract of hypertensive rats compared with normotensive rats, which was attributable to an increase in the proportion of dendritic spines containing GluR1 as well as an increase in the total number of dendritic spines. The differences were only seen after the development of hypertension and were not seen in rostral regions of the nucleus of the solitary tract. In the spontaneously hypertensive rat, many synapses on GluR1-containing dendrites had the morphological features of synapses undergoing dynamic changes, including the presence of perforated synapses. These results suggest that changes in afferent activity to the nucleus of the solitary tract during sustained hypertension alter both the dendritic structure and AMPA receptor content of some neurons. These structural changes may be a substrate for central resetting of the baroreceptor reflex.

Co-localization of AMPA receptor subunits in the nucleus of the solitary tract in the rat.

AMPA-type glutamate receptors in the caudal portions of nucleus of the solitary tract (NTS) are critical for responses to excitatory afferents from the viscera, including baroreceptors. Using immunocytochemistry combined with electron microscopy, the cellular distributions of different AMPA receptor subunits in the caudal NTS were found to be distinct. GluR2/3 was found at pre- and postsynaptic sites, and in astrocytic glia; while GluR1 was found primarily in small dendrites and spines. In dual-labeling studies, GluR1 and GluR2 were co-localized in large dendrites, but GluR1 was more often found alone in dendritic spines. Therefore, single neurons in the NTS contain both subunits, but there is differential trafficking of GluR1 to potential sites for synaptic plasticity.

Leptin potentiates thermogenic sympathetic responses to hypothermia: a receptor-mediated effect.

Leptin contributes to the regulation of thermogenesis. In rodents, sympathetic nerve activity efferent to interscapular brown adipose tissue (IBAT-SNA) is involved. On the basis of the hypotheses that 1) leptin acutely potentiates hypothermia-induced increases in IBAT-SNA; 2) this action of leptin is specific to IBAT-SNA, i.e., it does not occur with renal sympathetic nerve activity (R-SNA); and 3) this effect of leptin depends on intact and functional leptin receptors, we measured IBAT-SNA and R-SNA in anesthetized lean and diet-induced obese Sprague-Dawley and in obese Zucker rats, randomly assigned to low-dose leptin or vehicle. Before the start of leptin or vehicle and 5 min, 90 min, and 180 min after, hypothermia (30 degrees C) was induced. Compared with vehicle, leptin did not significantly alter baseline R-SNA or IBAT-SNA. In lean Sprague-Dawley rats, hypothermia-induced increases in IBAT-SNA were significantly augmented by leptin but not by vehicle. In obese Sprague-Dawley rats, leptin did not potentiate hypothermia-induced increases in IBAT-SNA. In Zucker rats, IBAT-SNA did not increase with hypothermia and leptin was not able to induce sympathoactivation with cooling. Changes in R-SNA during hypothermia were not significantly modified by leptin in either group. Thus, low-dose leptin, although not altering baseline SNA, acutely enhances hypothermia-induced sympathetic outflow to IBAT in lean rats. This effect is specific for thermogenic SNA because leptin does not significantly alter the response of R-SNA to hypothermia. The effect depends on intact and functional leptin receptors because it occurs neither in rats with a leptin receptor defect nor in rats with acquired leptin resistance.