Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes\' CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.

Bovine viral diarrhea virus\'s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

UNASSIGNED: Quantify impaired respiration in currently marketed crib bumpers (CBs), mesh liners (MLs) and alternative products (ALTs) used to attenuate the interaction between the baby and the crib sides and elucidate the relationship between impaired respiration and permeability.
UNASSIGNED: We experimentally quantified carbon dioxide rebreathing (CO2RB) via an infant manikin and air permeability via previously published test protocols, in commercially available CBs, MLs and ALTs.
UNASSIGNED: Differences in CO2RB in ML (median [m]=8.2%, 25th percentile [P25]=6.8, 75th percentile [P75]=8.6), ALT (m=10.5%, P25=9.8, P75=10.7) and CB (m=11.6%, P25=10.2, P75=14.3) were significant (p<0.0001). For comparison, manikin tests with a pacifier yielded CO2RB of 5.6%-5.9%, blanket draped over the face/torso yielded CO2RB of 7.7%-8.6% and stuffed animal in various positions yielded CO2RB from 6.1% to 16.1%. Differences in permeability between ML (m=529.5 cubic feet per minute [CFM], P25=460, P75=747.5), ALT (m=29.0 CFM, P25=27.7, P75=37.7) and CB (m=46.6 CFM, P25=30.1, P75=58.7) groups were significant (p<0.0001). CO2RB was poorly correlated with air permeability (max R2=0.36). In a subset of tests, CB CO2RB increased by 50%-80% with increasing penetration force, whereas the ML CO2RB was nominally unchanged.
UNASSIGNED: Government agencies and standards organisations are presently considering regulation of bedding including CBs. As paediatricians are consulted in the development of such regulations, our findings that permeability by itself was a poor predictor of CO2RB should be considered.

Little is known about whether sleep space impacts toddler sleep outcomes. We examined the prevalence of crib-sleeping and its association with caregiver-reported sleep patterns and problems in a large sample of toddlers from Western countries.
Participants were caregivers of 1983 toddlers ages 18.0-35.9 months (51.7% male; mean age 25.3 months) from Australia, Canada, New Zealand, the United Kingdom, and the United States sleeping in a crib or bed in a separate room from caregivers. Caregiver-reported sleep patterns and problems were collected via a free, publicly available child sleep smartphone application.
Across countries/regions, rates of crib-sleeping decreased linearly with age, with 63.4% of toddlers ages 18.0-23.9 months, 34.3% of toddlers ages 24.0-29.9 months, and 12.6% of toddlers ages 30.0-35.9 months sleeping in a crib. Across age groups and countries, crib sleeping was significantly associated with an earlier bedtime, shorter sleep onset latency, fewer night awakenings, longer stretches of time asleep, increased nighttime sleep duration, and decreased bedtime resistance and sleep problems. The duration of night awakenings did not significantly differ by sleep space.
Sleeping in a crib instead of a bed is associated with enhanced caregiver-reported sleep quantity and quality for toddlers in Western countries. Consistent with practice recommendations, deferring the crib-to-bed transition until age 3 years may benefit toddlers\' sleep in Western contexts. Additional research is needed to identify the impact of sleep space on child sleep in other countries/regions.

Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.

OBJECTIVE: Assess maternal psychological functioning within the Neonatal Intensive Care Unit (NICU) and its contribution to neonate length of stay (LOS) in the NICU.
METHODS: Mothers of infants admitted to the NICU (n=111) were assessed regarding postpartum depression, postpartum social support, postpartum NICU stress, and maternal anxiety at 2 weeks postpartum. Illness severity was assessed with the Clinical Risk Index for Babies (CRIB).
RESULTS: Postpartum depression was not significantly correlated with LOS, but was significantly correlated with trait anxiety (r=0.620), which was significantly correlated with LOS (r=0.227). Among mothers with previous mental health history, substance abuse history and CRIB score were the best predictors of LOS. For mothers without a prior mental health issues, delivery type, stress associated with infant appearance, and CRIB scores were the best predictors of LOS. In this group, LOS was found to increase on average by 7.06 days per one unit increase in stress associated with infant appearance among mothers with the same delivery type and CRIB score.
CONCLUSIONS: Significant correlations of trait anxiety, stress associated with infant appearance, and parental role with LOS support the tenet that postpartum psychological functioning can be associated with NICU LOS.

OBJECTIVE: To determine whether an early heart rate characteristics (HRC) index (HeRO score), measured in the first day and week after birth predicts death and morbidities compared with established illness severity scores.
METHODS: For all very low birth weight infants in a single neonatal intensive care unit from 2004-2014, the average first day HRC index was calculated within 24 hours of birth (aHRC-24h) and the average first week HRC index within 7 days of birth (aHRC-7d). The Score for Neonatal Acute Physiology (SNAP-II) and Clinical Risk Indicator for Babies (CRIB-II) were calculated when data were available. The aHRC was compared with the SNAP-II and CRIB-II for predicting death, late-onset septicemia, necrotizing enterocolitis, bronchopulmonary dysplasia, severe intraventricular hemorrhage, or severe retinopathy of prematurity.
RESULTS: All 4 scores were associated with death and severe intraventricular hemorrhage (P < .01). The OR and 95% CI for every 1-point increase in aHRC for predicting mortality, adjusted for gestational age, was 1.59 (1.25-2.00) for aHRC-24h and 2.61 (1.58-4.33) for aHRC-7d. High aHRC-7d, SNAP-II, and CRIB-II were associated with bronchopulmonary dysplasia (P < .001). High aHRC-7d was associated with late-onset septicemia (P < .05). None of the scores predicted necrotizing enterocolitis or severe retinopathy of prematurity.
CONCLUSIONS: HRC assessed in the first day or first week after birth compares favorably to established risk scores to predict death and morbidities in very low birth weight infants.

A quality improvement project for implementing safe sleep practices (SSP) was conducted at a large, U.S children\'s hospital. The intervention involved education of staff and standardization of infant sleep practices utilizing a multifaceted approach. Staff surveys and environmental audits were conducted pre- and post-intervention. Safe Sleep Environment (SSE) audits showed an improvement from 23% to 34% (p<0.001) post-intervention. Staff confidence to provide education to caregivers on SSP showed a significant increase. Results from this project demonstrate a successful approach to implement SSP in the hospital setting. Infant safe sleep practices have the potential to reduce infant mortality.

Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth.

BACKGROUND: Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP.
METHODS: The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3-/- mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3-/- mice.
RESULTS: The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3-/- mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3-/- mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3-/- mice-thereby confirming the role of MLK3 in neutrophil migration.
CONCLUSIONS: Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.