Wireless Fetal Heart Rate Monitoring in Inpatient Full-Term Pregnant Women: Testing Functionality and Acceptability

We tested functionality and acceptability of a wireless fetal monitoring prototype technology in pregnant women in an inpatient labor unit in the United States. Women with full-term singleton pregnancies and no evidence of active labor were asked to wear the prototype technology for 30 minutes. We assessed functionality by evaluating the ability to successfully monitor the fetal heartbeat for 30 minutes, transmit this data to Cloud storage and view the data on a web portal. Three obstetricians also rated fetal cardiotocographs on ease of readability. We assessed acceptability by administering closed and open-ended questions on perceived utility and likeability to pregnant women and clinicians interacting with the prototype technology. Thirty-two women were enrolled, 28 of whom (87.5%) successfully completed 30 minutes of fetal monitoring including transmission of cardiotocographs to the web portal. Four sessions though completed, were not successfully uploaded to the Cloud storage. Six non-study clinicians interacted with the prototype technology. The primary technical problem observed was a delay in data transmission between the prototype and the web portal, which ranged from 2 to 209 minutes. Delays were ascribed to Wi-Fi connectivity problems. Recorded cardiotocographs received a mean score of 4.2/5 (± 1.0) on ease of readability with an interclass correlation of 0.81(95%CI 0.45, 0.96). Both pregnant women and clinicians found the prototype technology likable (81.3% and 66.7% respectively), useful (96.9% and 66.7% respectively), and would either use it again or recommend its use to another pregnant woman (77.4% and 66.7% respectively). In this pilot study we found that this wireless fetal monitoring prototype technology has potential for use in a United States inpatient setting but would benefit from some technology changes. We found it to be acceptable to both pregnant women and clinicians. Further research is needed to assess feasibility of using this technology in busy inpatient settings.     

Is Early Tuberculosis Death Associated with Increased Tuberculosis Transmission?

Introduction

Tuberculosis (TB) is now a relatively uncommon disease in high income countries. As such, its diagnosis may be missed or delayed resulting in death before or shortly after the introduction of treatment. Whether early TB death is associated with increased TB transmission is unknown. To determine the transmission risk attributable to early TB death we undertook a case-control study.

Methods

All adults who were: (1) diagnosed with culture-positive pulmonary TB in the Province of Alberta, Canada between 1996 and 2012, and (2) died a TB-related death before or within the first 60 days of treatment, were identified. For each of these “cases” two sets of “controls” were randomly selected from among culture-positive pulmonary TB cases that survived beyond 60 days of treatment. “Controls” were matched by age, sex, population group, +/- smear status. Secondary cases of “cases” and “controls” were identified using conventional and molecular epidemiologic tools and compared. In addition, new infections were identified and compared in contacts of “cases” that died before treatment and contacts of their smear-matched “controls”. Conditional logistic regression was used to find associations in both univariate and multivariate analysis.

Results

“Cases” were as, but not more, likely than “controls” to transmit. This was so whether transmission was measured in terms of the number of “cases” and smear-unmatched or -matched “controls” that had a secondary case, the number of secondary cases that they had or the number of new infections found in contacts of “cases” that died before treatment and their smear-matched “controls”.

Conclusion

In a low TB incidence/low HIV prevalence country, pulmonary TB patients that die a TB-related death before or in the initial phase of treatment and pulmonary TB patients that survive beyond the initial phase of treatment are equally likely to transmit.     

A Cre‐inducible fluorescent reporter for observing apical membrane dynamics

The ability to image living tissues with fluorescent proteins has revolutionized the fields of cell and developmental biology. Fusions between fluorescent proteins and various polypeptides are allowing scientists to image tissues with sub‐cellular resolution. Here, we describe the generation and activity of a genetically engineered mouse line expressing a fusion between the green fluorescent protein (GFP) and the apically localized protein Crumbs3 (Crb3). This reporter drives Cre‐inducible expression of Crb3–GFP under control of the EF1a regulatory domains. The fusion protein is broadly expressed in embryonic and adult tissues and shows apical restriction in the majority of epithelial cell types. It displays a variably penetrant gain of function activity in the neural tube. However, in several cell types, over‐expression of Crb3 does not appear to have any effect on normal development or maintenance. Detailed analysis of kidneys expressing this reporter indicates normal morphology and function highlighting the utility for live imaging. Thus, the EF1a^Crb3–GFP mouse line will be of broad use for studying membrane and/or tissue dynamics in living tissues. genesis 53:285–293, 2015. © 2015 Wiley Periodicals, Inc.     

A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein–Peptide Array Screening

Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein–protein interactions in a systematic and reliable manner.Immune receptor signaling, critical for proper immune response, involves signaling pathways mediated by specific tyrosine residues that are phosphorylated upon receptor activation (1). These tyrosine phosphorylation sites are frequently found in one of the three types of immunoreceptor tyrosine-based regulatory motifs (ITRMs)¹. The immunoreceptor tyrosine-based activation motifs (ITAMs) with the consensus sequence YxxI/Lx(6–12)YxxI/L, where x represents any amino acid, are typically associated with positive or activating immune response (2). In contrast, the immune system elicits negative or inhibitory response through receptors bearing the immunoreceptor tyrosine-based inhibition motifs (ITIMs) with the degenerated sequence S/I/V/LxYxxI/V/L (3). Interestingly, SLAM/CD150 and related receptors of the CD2 subfamily contain a third type of signaling motif called Immunoreceptor Tyrosine-based Switch Motif (ITSM) with the consensus sequence TxYxx(V/I) (4). An ITSM can convey either an activating or inhibitory signal, depending on the type of immune cell, the receptor and the bound protein (5).In general, phosphorylated ITIMs recruit the SH2 domain-containing tyrosine phosphatase SHP-1 or SHP-2 (6), and phosphorylated ITAMs are recognized by protein tyrosine kinases such as ZAP70 in T cells and SYK in B cells (7). Nevertheless, these distinctions are only relative and both motifs may be involved in either positive or negative immune functions. For example, ITIM-containing inhibitory receptors found on phagocytes can suppress or enhance inflammatory cytokine production depending on the downstream proteins that they recruit (6). Similarly, ITAM sequences have been found to mediate inhibitory signaling (8). Indeed, even within the same cell type, an ITAM-containing receptor may mediate functions as diverse as microbial killing, antigen presentation, cytokine production, T-Cell instruction, and tissue repair (2). Moreover, exquisite binding specificities have been observed for different ITRMs. For example, the CD31 ITIMs recruit SHIP-1, SHP-1, and SHP-2 (9). In contrast, the archetypal ITIM identified in the cytoplasmic domain of the inhibitory IgG Fc receptor FcγRIIB (10) recruits SHIP-1 and -2, but not SHP-1 or -2. Although the FcγRIIB ITIM has an affinity for the SH2 domain of either SHP-1 or -2, it does not recruit these tyrosine phosphatases as do other ITIM-bearing receptors (11). Besides SHIP-1/2 and SHP-1/2, other SH2-containing proteins have also been found associated with certain ITIMs. For example, through their respective ITIM sequences, the LAIR-1 receptor recruits tyrosine kinase CSK (12), Lax interacts with GRB2, PIK3R1 and GRAP2 (13), and CD72 forms a complex with SHP-1 and GRB2 (14). These findings indicate that immunoreceptor signaling is context-dependent and that the precise pairing of an ITRM sequence with an SH2-containing protein plays a critical role in dictating the signaling and biological outcome.Given the critical importance of SH2 domain-phosphotyrosine (pY) interaction in immune signaling, we set to systematically identify SH2 domain-ITRM interactions. A variety of different high-throughput methods have been developed for the identification of protein–protein or protein-peptide interactions to date. These include yeast two-hybrid (15), affinity purification coupled to mass spectrometry (AP-MS) (16), phage display (17), and protein (18) and peptide arrays (19). We employed protein and peptide arrays in our study because of their simplicity and amenability to post-translational modifications. Specifically, we combined SH2 domain array with phosphotyrosine peptide array to decipher an SH2-ITRM interactome for immunoreceptors. We showed that the peptide and SH2 domain arrays were not simple mirror images of each other, but rather, they exhibited distinct, but complementary binding patterns. Although neither array platform alone was sufficient to detect all authentic interactions, integration of data from reciprocal peptide and protein array screens allowed us to identify a high-confidence SH2-ITRM interactome. The interactome has not only provided a systematic view of immunoreceptor signaling network, but also generated unique insights into the signaling specificity for the different immune signaling motifs.     

Limited phenological and pollinator-mediated isolation among selfing and outcrossing Arabidopsis lyrata populations

Transitions from outcrossing to selfing have been a frequent evolutionary shift in plants and clearly play a role in species divergence. However, many questions remain about the initial mechanistic basis of reproductive isolation during the evolution of selfing. For instance, how important are pre-zygotic pre-pollination mechanisms (e.g. changes in phenology and pollinator visitation) in maintaining reproductive isolation between newly arisen selfing populations and their outcrossing ancestors? To test whether changes in phenology and pollinator visitation isolate selfing populations of Arabidopsis lyrata from outcrossing populations, we conducted a common garden experiment with plants from selfing and outcrossing populations as well as their between-population hybrids. Specifically, we asked whether there was isolation between outcrossing and selfing plants and their between-population hybrids through differences in (1) the timing or intensity of flowering; and/or (2) pollinator visitation. We found that phenology largely overlapped between plants from outcrossing and selfing populations. There were also no differences in pollinator preference related to mating system. Additionally, pollinators preferred to visit flowers on the same plant rather than exploring nearby plants, creating a large opportunity for self-fertilization. Overall, this suggests that pre-zygotic pre-pollination mechanisms do not strongly reproductively isolate plants from selfing and outcrossing populations of Arabidopsis lyrata.     

GNAQQ209L expression initiated in multipotent neural crest cells drives aggressive melanoma of the central nervous system

Primary leptomeningeal melanocytic neoplasms represent a spectrum of rare tumors originating from melanocytes of the leptomeninges, which are the inner two membranes that protect the central nervous system. Like other non‐epithelial melanocytic lesions, they bear frequent oncogenic mutations in the heterotrimeric G protein alpha subunits, GNAQ or GNA11. In this study, we used Plp1‐creERT to force the expression of oncogenic GNAQ^Q209L in the multipotent neural crest cells of the ventro‐medial developmental pathway, beginning prior to melanocyte cell differentiation. We found that this produces leptomeningeal melanocytic neoplasms, including cranial melanocytomas, spinal melanocytomas, and spinal melanomas, in addition to blue nevus‐like lesions in the dermis. GNAQ^Q209L drove different phenotypes depending upon when during embryogenesis (E9.5, E10.5, or E11.5) it was induced by tamoxifen and which Cre driver (Plp1‐creERT, Tyr‐creERT², or Mitf‐cre) was used. Given these differences, we propose that melanocytes go through temporary phases where they become sensitive to the oncogenic effects of GNAQ^Q209L. R26‐fs‐GNAQ^Q209L; Plp1‐creERT mice will be useful for defining biomarkers for potentially aggressive leptomeningeal melanocytomas and for developing new therapeutics for advanced disease.     

γ-Ray-induced degradation of lignocellulosic materials

Lignocellulosic plant materials were treated with various swelling agents and exposed to γ radiation from ⁶⁰Co or ¹³⁷Cs. At dosages of 50 Mrad or above, lignocellulosic materials were extensively degraded and solubilized in water. Addition of water, NaOH, or H₂SO₄ to the substrate increased the degree of solubilization. Complete solubilization was achieved for samples of sugarcane bagasse, newspaper, cotton linters, cotton cloth, sawdust, and α-cellulose powder. About 35% total sugar and 5% reducing sugar per dry weight of sugarcane bagasse could be obtained by this method. Most of the soluble carbohydrates seemed to be disaccharides or larger molecules and glucose degradation products. Solubilization of cellulose was dosage dependent and although the rate of solubilization was increased by adding alkali, released sugar was further decomposed by the alkali and by high dosage of radiation.     

mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.     

Stressed and Depressed? Check Your GDNF for Epigenetic Repression

Some adults fail to adapt to chronic stress, developing symptoms of depression and anxiety. In this issue of Neuron, Uchida and colleagues link maladaptive stress responses to GDNF through a comprehensive investigation of the neurotrophic factor's regulation. Further, this study is an excellent example for investigators interested in neuroepigenetics research.     

Nodes of Ranvier act as barriers to restrict invasion of flanking paranodal domains in myelinated axons

Accumulation of voltage-gated sodium (Na(v)) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization, and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (Nfasc(NF1??)) in vivo results in nodal disorganization, including loss of Na(v) channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral nervous system (PNS) and central nervous system (CNS). Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in Nfasc(NF1??) mutant axons and occlude node formation. Our results suggest that Nfasc(NF1??)-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.