Serial exposure to ethanol drinking and methamphetamine enhances glutamate excitotoxicity.

A significant comorbidity exists between alcohol and methamphetamine (Meth) abuse but the neurochemical consequences of this co-abuse are unknown. Alcohol and Meth independently and differentially affect glutamatergic transmission but the unique effects of their serial exposure on glutamate signaling in mediating damage to dopamine neurons are unknown. Sprague-Dawley rats had intermittent voluntary access to 10% ethanol (EtOH) every other day and water over 28 days and were then administered a binge injection regimen of Meth or saline. EtOH drinking decreased the glutamate aspartate transporter and increased basal extracellular concentrations of glutamate within the striatum when measured after the last day of drinking. Ceftriaxone is known to increase the expression and/or activity of glutamate transporters in the brain and prevented both the decreases in glutamate aspartate transporter and the increases in basal extracellular glutamate when administered during EtOH drinking. EtOH drinking also exacerbated the acute increases in extracellular glutamate observed upon Meth exposure, the subsequent increases in spectrin proteolysis, and the long-term decreases in dopamine content in the striatum, all of which were attenuated by ceftriaxone administration during EtOH drinking only. These results implicate EtOH-induced increases in extracellular glutamate and corresponding decreases in glutamate uptake as mechanisms that contribute to the vulnerability produced by EtOH drinking and the unique neurotoxicity observed after serial exposure to Meth that is not observed with either drug alone. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Effect of Evidence-Based Practice (EBP) Courses on MSN and DNP Students' Use of EBP.

PURPOSE: The purpose of this study was to evaluate the effect of evidence-based practice (EBP) courses on nursing students' attitudes, perceived support from their professional network, self-efficacy, knowledge, and implementation of EBP. DESIGN: This study utilized a two-group, pre- and posttest design with 190 Masters of Science in Nursing (MSN) and 37 Doctorate of Nursing Practice (DNP) students. METHODS: An EBP instrument based on the theory of planned behavior was administered both before and after the EBP course (the intervention). FINDINGS: Both the pre- and posttest were completed by 126 students for a 56% response rate. No significant differences between the MSN (n = 102) and DNP (n = 24) students were found in precourse scores on any of the subscales except behavior, with the DNP students reporting they performed more EBP behaviors in the clinical setting. Overall, student scores on three of the four subscales of the EBP instrument (attitudes, self-efficacy, and behavior) significantly increased pre- to postcourse. The self-efficacy subscale demonstrated the greatest pre- to postcourse change scores. When the DNP and MSN students were compared, change scores on attitudes and self-efficacy remained significant in both groups. There was a significant positive change in EBP behavior only for the MSN students. Knowledge scores increased significantly only for the DNP students. LINKING EVIDENCE TO ACTION: EBP courses can increase self-reported EBP behaviors in the clinical setting, especially in MSN students. A precourse student evaluation will help faculty determine their learning needs in order to develop appropriate learning activities to support their acquisition of the essential knowledge, skills, and abilities to use EBP in the clinical setting. Administering the same evaluation postcourse can help faculty evaluate the effectiveness of their teaching. As a result, advanced practice nurses will be better equipped to facilitate and promote the implementation of EBP to support high-quality care and improved health outcomes.

Early skin-to-skin contact for mothers and their healthy newborn infants.

BACKGROUND: Mother-infant separation post birth is common. In standard hospital care, newborn infants are held wrapped or dressed in their mother's arms, placed in open cribs or under radiant warmers. Skin-to-skin contact (SSC) begins ideally at birth and should last continually until the end of the first breastfeeding. SSC involves placing the dried, naked baby prone on the mother's bare chest, often covered with a warm blanket. According to mammalian neuroscience, the intimate contact inherent in this place (habitat) evokes neuro-behaviors ensuring fulfillment of basic biological needs. This time frame immediately post birth may represent a 'sensitive period' for programming future physiology and behavior. OBJECTIVES: To assess the effects of immediate or early SSC for healthy newborn infants compared to standard contact on establishment and maintenance of breastfeeding and infant physiology. SEARCH METHODS: We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (17 December 2015), made personal contact with trialists, consulted the bibliography on kangaroo mother care (KMC) maintained by Dr Susan Ludington, and reviewed reference lists of retrieved studies. SELECTION CRITERIA: Randomized controlled trials that compared immediate or early SSC with usual hospital care. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trials for inclusion and risk of bias, extracted data and checked them for accuracy. Quality of the evidence was assessed using the GRADE approach. MAIN RESULTS: We included 46 trials with 3850 women and their infants; 38 trials with 3472 women and infants contributed data to our analyses. Trials took place in 21 countries, and most recruited small samples (just 12 trials randomized more than 100 women). Eight trials included women who had SSC after cesarean birth. All infants recruited to trials were healthy, and the majority were full term. Six trials studied late preterm infants (greater than 35 weeks' gestation). No included trial met all criteria for good quality with respect to methodology and reporting; no trial was successfully blinded, and all analyses were imprecise due to small sample size. Many analyses had statistical heterogeneity due to considerable differences between SSC and standard care control groups. Results for womenSSC women were more likely than women with standard contact to be breastfeeding at one to four months post birth, though there was some uncertainty in this estimate due to risks of bias in included trials (average risk ratio (RR) 1.24, 95% confidence interval (CI) 1.07 to 1.43; participants = 887; studies = 14; I² = 41%; GRADE: moderate quality). SSC women also breast fed their infants longer, though data were limited (mean difference (MD) 64 days, 95% CI 37.96 to 89.50; participants = 264; studies = six; GRADE:low quality); this result was from a sensitivity analysis excluding one trial contributing all of the heterogeneity in the primary analysis. SSC women were probably more likely to exclusively breast feed from hospital discharge to one month post birth and from six weeks to six months post birth, though both analyses had substantial heterogeneity (from discharge average RR 1.30, 95% CI 1.12 to 1.49; participants = 711; studies = six; I² = 44%; GRADE: moderate quality; from six weeks average RR 1.50, 95% CI 1.18 to 1.90; participants = 640; studies = seven; I² = 62%; GRADE: moderate quality).Women in the SCC group had higher mean scores for breastfeeding effectiveness, with moderate heterogeneity (IBFAT (Infant Breastfeeding Assessment Tool) score MD 2.28, 95% CI 1.41 to 3.15; participants = 384; studies = four; I² = 41%). SSC infants were more likely to breast feed successfully during their first feed, with high heterogeneity (average RR 1.32, 95% CI 1.04 to 1.67; participants = 575; studies = five; I² = 85%). Results for infantsSSC infants had higher SCRIP (stability of the cardio-respiratory system) scores overall, suggesting better stabilization on three physiological parameters. However, there were few infants, and the clinical significance of the test was unclear because trialists reported averages of multiple time points (standardized mean difference (SMD) 1.24, 95% CI 0.76 to 1.72; participants = 81; studies = two; GRADE low quality). SSC infants had higher blood glucose levels (MD 10.49, 95% CI 8.39 to 12.59; participants = 144; studies = three; GRADE: low quality), but similar temperature to infants in standard care (MD 0.30 degree Celcius (°C) 95% CI 0.13 °C to 0.47 °C; participants = 558; studies = six; I² = 88%; GRADE: low quality). Women and infants after cesarean birthWomen practicing SSC after cesarean birth were probably more likely to breast feed one to four months post birth and to breast feed successfully (IBFAT score), but analyses were based on just two trials and few women. Evidence was insufficient to determine whether SSC could improve breastfeeding at other times after cesarean. Single trials contributed to infant respiratory rate, maternal pain and maternal state anxiety with no power to detect group differences. SubgroupsWe found no differences for any outcome when we compared times of initiation (immediate less than 10 minutes post birth versus early 10 minutes or more post birth) or lengths of contact time (60 minutes or less contact versus more than 60 minutes contact). AUTHORS' CONCLUSIONS: Evidence supports the use of SSC to promote breastfeeding. Studies with larger sample sizes are necessary to confirm physiological benefit for infants during transition to extra-uterine life and to establish possible dose-response effects and optimal initiation time. Methodological quality of trials remains problematic, and small trials reporting different outcomes with different scales and limited data limit our confidence in the benefits of SSC for infants. Our review included only healthy infants, which limits the range of physiological parameters observed and makes their interpretation difficult.

Development and Validation of an Evidence-Based Practice Instrument for Nursing Students Based on the Theory of Planned Behavior.

BACKGROUND AND PURPOSE: This study developed and validated a theory of planned behavior (TPB)-based self-report instrument to measure nursing students' attitudes toward evidence-based practice (EBP), perceived support, self-efficacy, and implementation of EBP. METHODS: There were 348 nursing students at 1 university who completed the measure as a pretest at the beginning of a course designed to teach them about EBP; 164 at the end of the course as a posttest. RESULTS: Doctor of Nursing Practice (DNP) students reported higher EBP implementation scores than Master of Science in Nursing (MSN) students who, in turn, had higher scores than prespecialty students. At the pretest, self-efficacy and network support accounted for 31% of the variance in EBP implementation. CONCLUSIONS: Evidence provides initial support for the reliability and validity of this 4-part EBP instrument based on the TPB.

Reflections of Black Women Who Choose to Breastfeed: Influences, Challenges and Supports.

OBJECTIVES: Black women continue to have the lowest rates of breastfeeding. Of those who choose to breastfeed up to half cease nursing within the first few days or months postpartum. This study identified factors that influence and challenge Black women who choose to breastfeed, and supportive strategies that facilitate successful breastfeeding experiences. METHODS: Four focus groups were conducted in 2013 with 16 self-identified Black women aged 21-46 (M = 31.35 years), with 11-18 (M = 14.94) years of education, and were either pregnant or had given birth to an infant within the prior 5 years (range of pregnancies 1-7; M = 2.44). A standard set of questions guided discussions. Data saturation occurred after three groups. All discussions were audiotaped and transcribed. Qualitative methods were used to identify categories and subthemes. Reviewers met periodically to resolve ambiguities and coding discrepancies. Member checking was conducted. RESULTS: Four major categories emerged: Balancing the influences: People, myths, and technology; Being in the know; Critical periods; and, Supportive Transitions. Most women experienced little help with breastfeeding from health providers or systems. More influential was the interplay of family members,myths and the internet "as my friend". Role models and personalized support were noted as important but lacking among Black women. Patient profiling, experienced by some of the women, impacted breastfeeding choices. CONCLUSIONS: Black women such as our participants are critical partners as we develop systems of care to decrease disparities and increase Black women's successes in breastfeeding. Findings underscore the importance of having diverse, readily available, user-friendly, culturally sensitive options for Black women who choose to breastfeed.

The Effect of Directed Medical Play on Young Children's Pain and Distress During Burn Wound Care.

Directed medical play is used to reduce children's pain and distress during medical treatment. In this pilot study, young children who attended the burn clinic received either directed medical play provided by a child life specialist or standard preparation from the burn clinic nurse to prepare for their first dressing change. Data were collected using validated instruments. Children who participated in medical play experienced less distress during their dressing change (M = 0.5, n = 12) than did those receiving standard preparation (M = 2.0, n = 9). Children who received standard care reported a 2-point increase in pain during the procedure, whereas children who participated in medical play reported a 1-point increase. Change in parental anxiety was similar for both groups. Parent satisfaction was higher for caregivers who observed medical play than standard preparation. Although all findings were in the hypothesized direction, none was statically significant, most likely because of the small sample size.

Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins.

Chlamydia is an obligate intracellular pathogen that develops in the host cell in a vacuole termed the chlamydial inclusion. The prevailing concept of the chlamydial inclusion is of a parasitophorous vacuole. Here, the inclusion is the recipient of one-way host-pathogen interactions thus draining nutrients from the cell and negatively impacting it. While Chlamydia orchestrates some aspects of cell function, recent data indicate host cells remain healthy up until, and even after, chlamydial egress. Thus, while Chlamydia relies on the host cell for necessary metabolites, the overall function of the host cell, during chlamydial growth and development, is not grossly disturbed. This is consistent with the obligate intracellular organism's interest to maintain viability of its host. To this end, Chlamydia expresses inclusion membrane proteins, Incs, which serve as molecular markers for the inclusion membrane. Incs also contribute to the physical structure of the inclusion membrane and facilitate host-pathogen interactions across it. Given the function of Incs and the dynamic interactions that occur at the inclusion membrane, we propose that the inclusion behaves similarly to an organelle-albeit one that benefits the pathogen. We present the hypothesis that the chlamydial inclusion acts as a pathogen-specified parasitic organelle. This representation integrates the inclusion within existing subcellular trafficking pathways to divert a subset of host-derived metabolites thus maintaining host cell homeostasis. We review the known interactions of the chlamydial inclusion with the host cell and discuss the role of Inc proteins in the context of this model and how this perspective can impact the study of these proteins. Lessons learnt from the chlamydial pathogen-specified parasitic organelle can be applied to other intracellular pathogens. This will increase our understanding of how intracellular pathogens engage the host cell to establish their unique developmental niches.

The eukaryotic signal sequence, YGRL, targets the chlamydial inclusion.

Understanding how host proteins are targeted to pathogen-specified organelles, like the chlamydial inclusion, is fundamentally important to understanding the biogenesis of these unique subcellular compartments and how they maintain autonomy within the cell. Syntaxin 6, which localizes to the chlamydial inclusion, contains an YGRL signal sequence. The YGRL functions to return syntaxin 6 to the trans-Golgi from the plasma membrane, and deletion of the YGRL signal sequence from syntaxin 6 also prevents the protein from localizing to the chlamydial inclusion. YGRL is one of three YXXL (YGRL, YQRL, and YKGL) signal sequences which target proteins to the trans-Golgi. We designed various constructs of eukaryotic proteins to test the specificity and propensity of YXXL sequences to target the inclusion. The YGRL signal sequence redirects proteins (e.g., Tgn38, furin, syntaxin 4) that normally do not localize to the chlamydial inclusion. Further, the requirement of the YGRL signal sequence for syntaxin 6 localization to inclusions formed by different species of Chlamydia is conserved. These data indicate that there is an inherent property of the chlamydial inclusion, which allows it to recognize the YGRL signal sequence. To examine whether this "inherent property" was protein or lipid in nature, we asked if deletion of the YGRL signal sequence from syntaxin 6 altered the ability of the protein to interact with proteins or lipids. Deletion or alteration of the YGRL from syntaxin 6 does not appreciably impact syntaxin 6-protein interactions, but does decrease syntaxin 6-lipid interactions. Intriguingly, data also demonstrate that YKGL or YQRL can successfully substitute for YGRL in localization of syntaxin 6 to the chlamydial inclusion. Importantly and for the first time, we are establishing that a eukaryotic signal sequence targets the chlamydial inclusion.

Educating DNP students about critical appraisal and knowledge translation.

Consumers expect that health care providers will use the best evidence when assisting them in making decisions about treatment options. Nurses at all educational levels report that they lack knowledge to critically appraise research studies and the skills to effectively implement evidence-based practice (EBP) in their clinical settings. Organizational culture and management support of innovation are critical factors in the adoption of EBP. Doctor of Nursing Practice (DNP) graduates can have a pivotal role in the transfer of knowledge to practice, yet critical appraisal and EBP competencies for DNP and Master of Science in Nursing (MSN) students have not been well differentiated in nursing curricula. Also students' attitudes toward EBP, self-efficacy beliefs, utilization, and knowledge gaps are rarely evaluated before courses are designed. This article reports on the development of a DNP-level EBP course to help students evaluate and apply research findings to clinical practice.

Vesicle-associated membrane protein 4 and syntaxin 6 interactions at the chlamydial inclusion.

The predominant players in membrane fusion events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins. We hypothesize that SNARE proteins mediate fusion events at the chlamydial inclusion and are important for chlamydial lipid acquisition. We have previously demonstrated that trans-Golgi SNARE syntaxin 6 localizes to the chlamydial inclusion. To investigate the role of syntaxin 6 at the chlamydial inclusion, we examined the localization and function of another trans-Golgi SNARE and syntaxin 6-binding partner, vesicle-associated membrane protein 4 (VAMP4), at the chlamydial inclusion. In this study, we demonstrate that syntaxin 6 and VAMP4 colocalize to the chlamydial inclusion and interact at the chlamydial inclusion. Furthermore, in the absence of VAMP4, syntaxin 6 is not retained at the chlamydial inclusion. Small interfering RNA (siRNA) knockdown of VAMP4 inhibited chlamydial sphingomyelin acquisition, correlating with a log decrease in infectious progeny. VAMP4 retention at the inclusion was shown to be dependent on de novo chlamydial protein synthesis, but unlike syntaxin 6, VAMP4 recruitment is observed in a species-dependent manner. Notably, VAMP4 knockdown inhibits sphingomyelin trafficking only to inclusions in which it localizes. These data support the hypothesis that VAMP proteins play a central role in mediating eukaryotic vesicular interactions at the chlamydial inclusion and, thus, support chlamydial lipid acquisition and chlamydial development.

Sphingolipid trafficking and purification in Chlamydia trachomatis-infected cells.

Chlamydia trachomatis is an obligate intracellular human pathogen, which lacks a system that allows genetic manipulation. Therefore, chlamydial researchers must manipulate the host cell to better understand chlamydial biology. Host-derived lipid acquisition is critical for chlamydial survival within the host. Hence, the ability to track and purify sphingolipids in/from chlamydial infected cells has become an integral part of pivotal studies in chlamydial biology. This unit outlines protocols that provide details about labeling eukaryotic cells with exogenous lipids to examine Golgi-derived lipid trafficking to the chlamydial inclusion and then performing imaging studies or lipid extractions for quantification. Details are provided to allow these protocols to be applied to subconfluent, polarized, or siRNA knockdown cells. In addition, one will find important experimental design considerations and techniques. These methods are powerful tools to aid in the understanding of mechanisms, which allow C. trachomatis to manipulate and usurp host cell trafficking pathways.

Examining the role of actin-plasma membrane association in Pseudomonas aeruginosa infection and type III secretion translocation in migratory T24 epithelial cells.

The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.

Early skin-to-skin contact for mothers and their healthy newborn infants.

BACKGROUND: Mother-infant separation postbirth is common in Western culture. Early skin-to-skin contact (SSC) begins ideally at birth and involves placing the naked baby, head covered with a dry cap and a warm blanket across the back, prone on the mother's bare chest. According to mammalian neuroscience, the intimate contact inherent in this place (habitat) evokes neurobehaviors ensuring fulfillment of basic biological needs. This time may represent a psychophysiologically 'sensitive period' for programming future physiology and behavior. OBJECTIVES: To assess the effects of early SSC on breastfeeding, physiological adaptation, and behavior in healthy mother-newborn dyads. SEARCH METHODS: We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (30 November 2011), made personal contact with trialists, and consulted the bibliography on kangaroo mother care (KMC) maintained by Dr. Susan Ludington. SELECTION CRITERIA: Randomized controlled trials comparing early SSC with usual hospital care. DATA COLLECTION AND ANALYSIS: We independently assessed trial quality and extracted data. Study authors were contacted for additional information. MAIN RESULTS: Thirty-four randomized controlled trials were included involving 2177 participants (mother-infant dyads). Data from more than two trials were available for only eight outcome measures. For primary outcomes, we found a statistically significant positive effect of early SSC on breastfeeding at one to four months postbirth (13 trials; 702 participants) (risk ratio (RR) 1.27, 95% confidence interval (CI) 1.06 to 1.53, and SSC increased breastfeeding duration (seven trials; 324 participants) (mean difference (MD) 42.55 days, 95% CI -1.69 to 86.79) but the results did not quite reach statistical significance (P = 0.06). Late preterm infants had better cardio-respiratory stability with early SSC (one trial; 31 participants) (MD 2.88, 95% CI 0.53 to 5.23). Blood glucose 75 to 90 minutes following the birth was significantly higher in SSC infants (two trials, 94 infants) (MD 10.56 mg/dL, 95% CI 8.40 to 12.72).The overall methodological quality of trials was mixed, and there was high heterogeneity for some outcomes. AUTHORS' CONCLUSIONS: Limitations included methodological quality, variations in intervention implementation, and outcomes. The intervention appears to benefit breastfeeding outcomes, and cardio-respiratory stability and decrease infant crying, and has no apparent short- or long-term negative effects. Further investigation is recommended. To facilitate meta-analysis, future research should be done using outcome measures consistent with those in the studies included here. Published reports should clearly indicate if the intervention was SSC with time of initiation and duration and include means, standard deviations and exact probability values.

The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusion is isolated from the endocytic pathway but fusogenic with Golgi-derived exocytic vesicles containing sphingomyelin and cholesterol. Sphingolipids are incorporated into the chlamydial cell wall and are considered essential for chlamydial development and viability. The mechanisms by which chlamydiae obtain eukaryotic lipids are poorly understood but require chlamydial protein synthesis and presumably modification of the inclusion membrane to initiate this interaction. A polarized cell model of chlamydial infection has demonstrated that chlamydiae preferentially intercept basolaterally directed, sphingomyelin-containing exocytic vesicles. Here we examine the localization and potential function of trans-Golgi and/or basolaterally associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in chlamydia-infected cells. The trans-Golgi SNARE protein syntaxin 6 is recruited to the chlamydial inclusion in a manner that requires chlamydial protein synthesis and is conserved among all chlamydial species examined. The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma membrane retrieval signal (YGRL). Thus in addition to expression of at least two inclusion membrane proteins that contain SNARE-like motifs, chlamydiae also actively recruit eukaryotic SNARE-family proteins.

Role of host cell polarity and leading edge properties in Pseudomonas type III secretion.

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.

The chlamydial inclusion preferentially intercepts basolaterally directed sphingomyelin-containing exocytic vacuoles.

Chlamydiae replicate intracellularly within a unique vacuole termed the inclusion. The inclusion circumvents classical endosomal/lysosomal pathways but actively intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To further examine this interaction, we developed a polarized epithelial cell model to study vectoral trafficking of lipids and proteins to the inclusion. We examined seven epithelial cell lines for their ability to form single monolayers of polarized cells and support chlamydial development. Of these cell lines, polarized colonic mucosal C2BBe1 cells were readily infected with Chlamydia trachomatis and remained polarized throughout infection. Trafficking of (6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)hexanoyl)sphingosine) (NBD-C(6)-ceramide) and its metabolic derivatives, NBD-glucosylceramide (GlcCer) and NBD-SM, was analyzed. SM was retained within L2-infected cells relative to mock-infected cells, correlating with a disruption of basolateral SM trafficking. There was no net retention of GlcCer within L2-infected cells and purification of C. trachomatis elementary bodies from polarized C2BBe1 cells confirmed that bacteria retained only SM. The chlamydial inclusion thus appears to preferentially intercept basolaterally-directed SM-containing exocytic vesicles, suggesting a divergence in SM and GlcCer trafficking. The observed changes in lipid trafficking were a chlamydia-specific effect because Coxiella burnetii-infected cells revealed no changes in GlcCer or SM polarized trafficking.

Randomized controlled trial of very early mother-infant skin-to-skin contact and breastfeeding status.

This study was done to evaluate effects of maternal-infant skin-to-skin contact during the first 2 hours postbirth compared to standard care (holding the infant swaddled in blankets) on breastfeeding outcomes through 1 month follow-up. Healthy primiparous mother-infant dyads were randomly assigned by computerized minimization to skin-to-skin contact (n = 10) or standard care (n = 10). The Infant Breastfeeding Assessment Tool was used to measure success of first breastfeeding and time to effective breastfeeding (time of the first of three consecutive scores of 10-12). Intervention dyads experienced a mean of 1.66 hours of skin-to-skin contact. These infants, compared to swaddled infants, had higher mean sucking competency during the first breastfeeding (8.7 +/- 2.1 vs 6.3 +/- 2.6; P < .02) and achieved effective breastfeeding sooner (935 +/- 721 minutes vs 1737 +/- 1001; P < .04). No significant differences were found in number of breastfeeding problems encountered during follow-up (30.9 +/- 5.51 vs 32.7 +/- 5.84; P < .25) or in breastfeeding exclusivity (1.50 +/- 1.1 vs 2.10 +/- 2.2; P < .45). Sucking competency was also related to maternal nipple protractility (r = .48; P < .03). Very early skin-to-skin contact enhanced breastfeeding success during the early postpartum period. No significant differences were found at 1 month.

Prenatal and postpartum focus groups with primiparas: breastfeeding attitudes, support, barriers, self-efficacy, and intention.

INTRODUCTION: The purpose of this study was to explore women's prenatal attitudes, perception of support, anticipated barriers, facilitators, and breastfeeding self-efficacy beliefs and how their attitudes, beliefs, and perceptions of support changed as a result of their postpartum experiences. METHODS: A prospective, descriptive design with qualitative data collection methods was used for this study. Eight primiparas participated in prenatal and postpartum focus groups held in the conference room of a local school of nursing. One additional mother participated in individual interviews before and after giving birth at her request. RESULTS: In the prenatal groups, the major themes included beliefs that breastfeeding benefits both the mother and baby, availability of support, looking toward the future, and uncertainty about what to expect with breastfeeding. In the postpartum groups, the major themes centered around the realization that breastfeeding was both easy and difficult, the importance and role of supportive others, receiving conflicting advice, having validating experiences, and modifying breastfeeding intention based on postpartum experiences. DISCUSSION: Mothers need to be better educated for breastfeeding prenatally, and the information must be consistent, realistic, and evidence-based.