Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates

Allowing Relationships to Unfold: Consult Reason and Topics Discussed in Initial and Subsequent Palliative Care Visits Among Children Who Died From Relapsed/Refractory Cancer.

Background: Children with relapsed/refractory cancer have a myriad of palliative care needs. While pediatric oncology clinicians meet many of these needs, studies suggest that these children often have distressing symptoms and that families feel unprepared for their childs end-of-life (EOL). Oncology clinicians cite barriers to pediatric palliative care (PPC) consultation, including concerns that PPC teams will upset families with EOL discussions. This study evaluated topics addressed by PPC teams over the course of their relationship with children who died from cancer. Methods: Retrospective chart review of children who were diagnosed with relapsed/refractory cancer, received PPC consultation at an academic childrens hospital, and died between January 2008 and January 2017. Information was extracted regarding the childs treatment, EOL care, and the content of PPC consultation over the course of the teams relationship with the child/family. Results: Fifty-six children were included in the analysis. The most frequent reasons for the initial consult were pain (n = 31, 55%) and non-pain symptom management (n = 18, 32%). At the initial consult, the PPC team most often discussed symptom management and psychosocial support. Prognosis was not discussed in any initial consult. Over subsequent visits, the PPC team expanded their scope of discussion to include goals of care, advance care planning, and hospice. Discussion: Concerns from oncology clinicians that PPC teams will extend beyond the reasons for initial consult into prognostic/EOL discussions at the first visit may be unfounded. Greater familiarity with PPC team practices may facilitate more timely consultation of PPC and its complementary set of services

Inhibition of different luciferase transcriptional reporters by YopH deletion mutants.

<p>A, Activation of a <i>luciferase</i> reporter gene driven by NFAT/AP1. B, Activation of a NF-κB-driven <i>luciferase</i> reporter gene. C, Activation of a <i>luciferase</i> reporter gene driven by IL-2 promoter. In all cases, the data represent the mean±S.D. from triplicate determinations. *, p<0.05, **, p<0.005, ***, p<0.0005 as compared with pEF5 trasfected cells stimulated through TCR and CD28.</p

Pull-down assays with different deletion mutants of YopH.

<p>A, Schematic diagram showing the different deletion mutants of YopH used in this study. B, HEK293 cells expressing different proteins and treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed were lysed and probed for interaction with GST-YopH D/A (mutation D346A), the different deletion mutants shown in A fused to GST, and GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with YopH fragments was detected by Western blot with specific antibodies for Lck and Vav, and with anti-HA antibody for other proteins. An independent experiment was done for each protein. The lower panel shows a representative blot from one of the experiments to show that similar amounts of GST proteins were used in these assays.</p

Goals of Care Among Parents of Children Receiving Palliative Care

IMPORTANCE: While knowing the goals of care (GOCs) for children receiving pediatric palliative care (PPC) are crucial for guiding the care they receive, how parents prioritize these goals and how their priorities may change over time is not known. OBJECTIVE: To determine parental prioritization of GOCs and patterns of change over time for parents of children receiving palliative care. DESIGN, SETTING, AND PARTICIPANTS: A Pediatric Palliative Care Research Network\u27s Shared Data and Research cohort study with data collected at 0, 2, 6, 12, 18, and 24 months in hospital, outpatient, or home settings from April 10, 2017, to February 15, 2022, at 7 PPC programs based at children\u27s hospitals across the US. Participants included parents of patients, birth to 30 years of age, who received PPC services. EXPOSURES: Analyses were adjusted for demographic characteristics, number of complex chronic conditions, and time enrolled in PPC. MAIN OUTCOMES: Parents\u27 importance scores, as measured using a discrete choice experiment, of 5 preselected GOCs: seeking quality of life (QOL), health, comfort, disease modification, or life extension. Importance scores for the 5 GOCs summed to 100. RESULTS: A total of 680 parents of 603 patients reported on GOCs. Median patient age was 4.4 (IQR, 0.8-13.2) years and 320 patients were male (53.1%). At baseline, parents scored QOL as the most important goal (mean score, 31.5 [SD, 8.4]), followed by health (26.3 [SD, 7.5]), comfort (22.4 [SD, 11.7]), disease modification (10.9 [SD, 9.2]), and life extension (8.9 [SD, 9.9]). Importantly, parents varied substantially in their baseline scores for each goal (IQRs more than 9.4), but across patients in different complex chronic conditions categories, the mean scores varied only slightly (means differ 8.7 or less). For each additional study month since PPC initiation, QOL was scored higher by 0.06 (95% CI, 0.04-0.08) and comfort scored higher by 0.3 (95% CI, 0-0.06), while the importance score for life extension decreased by 0.07 (95% CI, 0.04-0.09) and disease modification by 0.02 (95% CI, 0-0.04); health scores did not significantly differ from PPC initiation. CONCLUSIONS AND RELEVANCE: Parents of children receiving PPC placed the highest value on QOL, but with considerable individual-level variation and substantial change over time. These findings emphasize the importance of reassessing GOCs with parents to guide appropriate clinical intervention

Pull-down assays with GST-YopH D356A.

<p>A, HEK293 cells expressing different proteins, either untreated (control) or treated with pervanadate (PV) to induce tyrosine phosphorylation of the proteins expressed, were lysed and probed for interaction with GST-YopH D346A or GST (5 µg each) as a negative control in pull-down assays. The specific interaction of those proteins with GST-YopH D356A was detected by Western blot with specific antibodies for Lck or Vav, and with anti-HA antibody for other proteins. B, As in A, HEK293 cells, expressing the same proteins and treated with PV, were lysed and probed for interaction with two different amounts of GST-YopH D346A (5 and 2 µg) or GST (5 µg each) as a negative control in pull-down assays. GST and GST-YopH D346A fusion protein used in these assays are shown at the lower panel from one representative blot. TL denotes total lysates of the transfected cells and corresponds to a 10% of the amount used for each pull-down assay. Assays were done independently for each protein.</p