Adapted Helping Babies Breathe approach to neonatal resuscitation in Haiti: a retrospective cohort study.

BACKGROUND: Helping Babies Breathe (HBB) is an American Academy of Pediatrics neonatal resuscitation program designed to reduce neonatal mortality in low resource settings. The 2017 neonatal mortality rate in Haiti was 28 per 1000 live births and an estimated 85 % of Haitian women deliver at home. Given this, the Community Health Initiative implemented an adapted HBB (aHBB) in Haiti to evaluate neonatal mortality. METHODS: Community Health Workers taught an aHBB program to laypeople, which didn't include bag-valve-mask ventilation. Follow-up after delivery assessed for maternal and neonatal mortality and health. RESULTS: Analysis included 536 births of which 84.3 % (n=452) were attended by someone trained in aHBB. The odds of neonatal mortality was not significantly different among the two groups (aOR=0.48 [0.16-1.44]). Composite outcome of neonatal health as reported by the mother (subjective morbidity and mortality) was significantly lower in aHBB attended births (aOR=0.31 [0.14-0.70]). CONCLUSION: This analysis of the aHBB program indicates that community training to laypersons in low resource settings may reduce neonatal ill-health but not neonatal mortality. This study is likely underpowered to find a difference in neonatal mortality. Further work is needed to evaluate which components of the aHBB program are instrumental in improving neonatal health.

The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the bacterial protein interferes with keratinocyte migration and proliferation.

Calcium microdomains at the immunological synapse: how ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation.

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.

Syntaxin7 is required for lytic granule release from cytotoxic T lymphocytes.

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL-mediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.

Docking of lytic granules at the immunological synapse in human CTL requires Vti1b-dependent pairing with CD3 endosomes.

Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8(+) T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.

The immunological synapse controls local and global calcium signals in T lymphocytes.

Cell polarization is a key feature of T-cell function. The immunological synapse (IS) between T cells and antigen-presenting cells is a beautiful example of how polarization of cells is used to guide cell function. Receptors, signal transducers, the cytoskeleton, and organelles are enriched at or depleted from the IS after its formation, and in many cases these re-localizations have already been linked with certain T-cell functions. One key step for T-cell activation is a rise in the cytoplasmic calcium concentration. Whereas it is undisputed that the IS initiates and controls calcium signals in T cells, very little is known about the role of T-cell polarization for calcium signals and calcium-dependent signal transduction. We briefly summarize the basic commonly agreed principles of IS-dependent calcium signal generation but then focus on the less well understood influence of polarization on calcium signals. The discussion of the role of polarization for calcium signals leads to a model how the IS controls local and global calcium signals and calcium-dependent T-cell functions. We develop a theoretical formalism based on existing spatiotemporal calcium dynamic simulations to better understand the model in the future and allow further predictions which can be tested by fast, high resolution live-cell microscopy.

Clinical Ultrasound Training in Emergency Medicine Advanced Practice Provider Residencies.

INTRODUCTION: Clinical ultrasound training is essential to any emergency medicine (EM) clinician's skill set. We aim to understand the current training patterns of clinical ultrasound training within Advanced Practice Provider (APP) residencies. METHODS: In a survey sent electronically to 17 active EM APP residencies, data were obtained from 21 responses to questions about structure of ultrasound faculty, quality assessment, feedback, and competency evaluation. RESULTS: We had a response rate of 88%. Of programs surveyed, 93% were associated with EM physician residencies with 87% led by an ultrasound fellowship-trained EM physician. Ninety-three percent of programs required proctored scanning. Sixty percent of programs do not have any required number of scans to graduate. CONCLUSION: We found that most EM APP residencies share clinical ultrasound faculty, structures, and processes with associated EM physician residencies. We believe that quality training within clinical ultrasound is attainable; however, proficiency guidelines across EM APP residency programs are lacking.

Pediatric Residents' Perceptions of a Point-of-Care Ultrasound Collaboration With Emergency Medicine.

Background Pediatric residencies expanding their point-of-care ultrasound (POCUS) education face barriers, including a lack of established curriculum and qualified educators. Prior studies report partnerships between pediatrics and pediatric emergency medicine (PEM); however, many non-PEM emergency medicine (EM) physicians with POCUS fellowship training also have experience with pediatric POCUS and represent an alternate educational partner. Objectives To improve pediatric residents' POCUS skills through collaborative education with EM and evaluate perceptions of the teaching format and instructors. Methods First through third-year pediatric residents attended a half-day didactic and hands-on session about renal, lung, and musculoskeletal (MSK) POCUS. These educational sessions were led by EM faculty with POCUS fellowship training and assisted by EM residents. Post-session surveys were administered to pediatric residents to assess prior POCUS experience, changes in confidence in acquiring and interpreting renal, lung, and MSK POCUS images, and opinions about the educational format. Statistical analyses of the post-session survey data were performed using SPSS. Results Thirty-nine pediatric residents attended the session and completed the survey of 45 total residents in the program (86.7%), with 89.7% completing 10 or fewer POCUS studies. Residents' comfort level with performing lung POCUS increased from 5.1% to 82.1% (p < .001), renal POCUS from 10.3% to 76.9% (p < .001), and MSK POCUS from 7.7% to 84.6% (p < .001). 87.2% rated the educational format as effective, and 94.9% (37/39) rated emergency medicine faculty as 'very effective' in providing ultrasound education relevant to the practice of pediatrics. Conclusion Pediatric resident POCUS education taught by EM faculty with POCUS fellowship training was well-received by pediatric residents and significantly improved confidence in acquiring and interpreting POCUS.

Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals.

Sustained Ca(2+) influx through plasma membrane Ca(2+) released-activated Ca(2+) (CRAC) channels is essential for T cell activation. Since inflowing Ca(2+) inactivates CRAC channels, T cell activation is only possible if Ca(2+)-dependent inactivation is prevented. We have previously reported that sustained Ca(2+) influx through CRAC channels requires both mitochondrial Ca(2+) uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca(2+)-dependent channel inactivation. Here, we show that morphological changes following formation of the immunological synapse (IS) modulate Ca(2+) influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca(2+) entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca(2+) signals. The shape change-dependent mitochondrial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca(2+) influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca(2+) dependent T cell activation.