A phage satellite manipulates the viral DNA packaging motor to inhibit phage and promote satellite spread.

ICP1, a lytic bacteriophage of Vibrio cholerae, is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs' dependence on ICP1's DNA replication machinery, and virion components results in inhibition of ICP1's lifecycle. PLEs' are expected to depend on ICP1 factors for genome packaging, but the mechanism(s) PLEs use to inhibit ICP1 genome packaging is currently unknown. Here, we identify and characterize Gpi, PLE's indiscriminate genome packaging inhibitor. Gpi binds to ICP1's large terminase (TerL), the packaging motor, and blocks genome packaging. To overcome Gpi's negative effect on TerL, a component PLE also requires, PLE uses two genome packaging specifiers, GpsA and GpsB, that specifically allow packaging of PLE genomes. Surprisingly, PLE also uses mimicry of ICP1's pac site as a backup strategy to ensure genome packaging. PLE's pac site mimicry, however, is only sufficient if PLE can inhibit ICP1 at other stages of its lifecycle, suggesting an advantage to maintaining Gpi, GpsA, and GpsB. Collectively, these results provide mechanistic insights into another stage of ICP1's lifecycle that is inhibited by PLE, which is currently the most inhibitory of the documented phage satellites. More broadly, Gpi represents the first satellite-encoded inhibitor of a phage TerL.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, PLE, remodels the capsid it has been predicted to steal from the phage ICP1 (1). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like-particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold, that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (2). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

Identification and development of a subtype-selective allosteric AKT inhibitor suitable for clinical development.

The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.

Bacteriophage ICP1: A Persistent Predator of Vibrio cholerae.

Bacteriophages or phages-viruses of bacteria-are abundant and considered to be highly diverse. Interestingly, a particular group of lytic Vibrio cholerae-specific phages (vibriophages) of the International Centre for Diarrheal Disease Research, Bangladesh cholera phage 1 (ICP1) lineage show high levels of genome conservation over large spans of time and geography, despite a constant coevolutionary arms race with their host. From a collection of 67 sequenced ICP1 isolates, mostly from clinical samples, we find these phages have mosaic genomes consisting of large, conserved modules disrupted by variable sequences that likely evolve mostly through mobile endonuclease-mediated recombination during coinfection. Several variable regions have been associated with adaptations against antiphage elements in V. cholerae; notably, this includes ICP1's CRISPR-Cas system. The ongoing association of ICP1 and V. cholerae in cholera-endemic regions makes this system a rich source for discovery of novel defense and counterdefense strategies in bacteria-phage conflicts in nature.

A phage satellite tunes inducing phage gene expression using a domesticated endonuclease to balance inhibition and virion hijacking.

Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1's capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.

How do new/recent mothers experience unwanted harm thoughts related to their newborn? A thematic analysis.

Objective: The aim of this qualitative research study was to explore how new/recent mothers experience intrusive, infant-related harm thoughts (IRHTs). Background: New/recent mothers' experiences of IRHTs remain a taboo topic, yet several studies indicate such thoughts are common. Understanding the normal experience of such thoughts is needed to improve the postpartum experiences of mothers, and clinical practice. The aim was to elicit a fuller, critical understanding of the experience of harm thoughts in a mixed, non-clinical sample, exploring how they are understood, managed and shared by women. Methods: Semi-structured interviews were conducted with eight new/recent mothers who had experienced IRHTs related to their youngest child, born within the last two years. Data were analysed with Thematic Analysis. Findings: The results yielded three core themes: Heightened emotions; Constructions of motherhood and maternal identity; Costs and benefits of sharing. Conclusion: Findings corroborate previous literature showing that IRHTs commonly occur among non-clinical postpartum mothers, highlighting their intense emotional impact, and barriers to women reporting them due to stigma. Results also highlighted ways in which the pervasive ideology of motherhood informed mothers' meaning-making of IRHTs. Recommendations include discussing the ideology of motherhood with pregnant women and partners and facilitating open, normalising discussion of IRHTs.

Prevalence and predictive factors of psychological morbidity following facial injury: a prospective study of patients attending a maxillofacial outpatient clinic within a major UK city.

Adults presenting to maxillofacial surgery services are at high risk of psychological morbidity. This study examined the prevalence of depression, post-traumatic stress disorder (PTSD), anxiety, drug and alcohol use, and appearance-related distress among maxillofacial trauma outpatients over medium-term follow-up. It also explored socio-demographic and injury-related variables associated with psychological distress to inform targeted psychological screening protocols for maxillofacial trauma services. Significant associations were found between level of distress at time of injury and number of traumatic life events with levels of depression at 3 months. No significant associations were found between predictor variables and PTSD at 3 months, or with any psychiatric diagnosis at 6 months. The lack of evidence for an identifiable subgroup of patients who were at higher risk of psychological distress indicated that routine screening of all maxillofacial trauma outpatients should be offered in order to best respond to their mental health needs. The feasibility of the medical team facilitating this is challenging and should ideally be undertaken by psychologists integrated within the MDT. This study led to the funding of a clinical psychologist to provide collaborative care with the maxillofacial surgeons, resulting in brief assessment and treatment to over 600 patients in the first year of the service.

Los adultos que consultan en los servicios de cirugía máxilo-facial tienen un alto riesgo de presentar morbilidad psicológica. Este estudio examinó la prevalencia de depresión, trastorno por estrés postraumático (TEPT), ansiedad, uso de alcohol y drogas, y distrés relacionado con la apariencia entre los pacientes con trauma máxilofacial en un seguimiento ambulatorio de mediano plazo. También se exploraron variables socio-demográficas y otras relacionadas con las lesiones que se asocian con distrés psicológico para contar con protocolos de evaluación psicológica orientados a los servicios de trauma máxilo-facial. Se encontraron asociaciones significativas entre el nivel de distrés al momento de la lesión y el número de acontecimientos traumáticos con los niveles de depresión a los tres meses. En cambio, no hubo asociaciones significativas entre las variables predictoras y el TEPT a los tres meses, o con algún diagnóstico psiquiátrico a los seis meses. La falta de evidencia de un subgrupo identificable de pacientes que estuvieron en alto riesgo de distrés psicológico indicaron que se debe ofrecer la evaluación de rutina a todos los pacientes ambulatorios con trauma máxilo-facial para responder mejor a sus necesidades de salud mental. Constituye un desafío configurar un equipo médico que permita esto y lo ideal es que se forme un equipo multidisciplinario en que estén integrados psicólogos. Este estudio permitió el financiamiento de un psicólogo clínico, quien aportó atención en colaboración con los cirujanos máxilo-faciales, lo que se tradujo en una evaluación breve y el tratamiento de más de 600 pacientes durante el primer año de funcionamiento del servicio.

Les adultes hospitalisés des services de chirurgie maxillo-faciale sont à risque élevé de morbidité psychologique. Cette étude analyse la prévalence de la dépression, du syndrome de stress post-traumatique (SSPT), de l'anxiété, de la consommation de drogues et d'alcool ainsi que de la détresse liée à l'apparence chez des patients ayant subi un traumatisme maxillo-facial, avec un suivi à moyen terme en ambulatoire. Les variables socio-démographiques et liées à la lésion ainsi que la détresse psychologique sont également examinées afin de renseigner des protocoles ciblés de dépistage psychologique pour les services de chirurgie maxillo-faciale. Le niveau de détresse au moment de la lésion et le nombre d'événements traumatiques de la vie sont significativement associés aux niveaux de dépression à 3 mois. Aucune association significative n'a été trouvée entre les variables prédictives et le SSPT à 3 mois ou un diagnostic psychiatrique quel qu'il soit à 6 mois. L'identification d'un sous-groupe de patients à risque élevé de détresse psychologique est difficile : le dépistage de routine de tous les patients suivis en ambulatoire après chirurgie maxillo-faciale devrait donc être proposé afin de mieux répondre à leurs besoins en santé mentale. Créer l'équipe médicale qui le permettrait est compliqué ; idéalement, cette tâche devrait être confiée à des psychologues au sein d'une équipe pluridisciplinaire. Grâce à l'étude, le poste d'un psychologue clinicien a été financé, qui travaille en collaboration avec les chirurgiens maxillo-faciaux. C'est ainsi que plus de 600 patients ont été évalués et traités au cours de la première année.

The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi.

Here we present an examination of type IV pilus genes associated with competence and twitching in the bacterium Acinetobacter baylyi (strain ADP1, BD413). We used bioinformatics to identify potential competence and twitching genes and their operons. We measured the competence and twitching phenotypes of the bioinformatically-identified genes. These results demonstrate that competence and twitching in A. baylyi both rely upon a core of the same type IV pilus proteins. The core includes the inner membrane assembly platform (PilC), a periplasmic assemblage connecting the inner membrane assembly platform to the secretin (ComM), a secretin (ComQ) and its associated pilotin (PilF) that assists with secretin assembly and localization, both cytoplasmic pilus retraction ATPases (PilU, PilT), and pilins (ComP, ComB, PilX). Proteins not needed for both competence and twitching are instead found to specialize in either of the two traits. The pilins are varied in their specialization with some required for either competence (FimT) and others for twitching (ComE). The protein that transports DNA across the inner membrane (ComA) specializes in competence, while signal transduction proteins (PilG, PilS, and PilR) specialize in twitching. Taken together our results suggest that the function of accessory proteins should not be based on homology alone. In addition the results suggest that in A. baylyi the mechanisms of natural transformation and twitching are mediated by the same set of core Type IV pilus proteins with distinct specialized proteins required for each phenotype. Finally, since competence requires multiple pilins as well as both pilus retraction motors PilU and PilT, this suggests that A. baylyi employs a pilus in natural transformation.

Succinate, iron chelation, and monovalent cations affect the transformation efficiency of Acinetobacter baylyi ATCC 33305 during growth in complex media.

Natural transformation is the acquisition of new genetic material via the uptake of exogenous DNA by competent bacteria. Acinetobacter baylyi is model for natural transformation. Here we focus on the natural transformation of A. baylyi ATCC 33305 grown in complex media and seek environmental conditions that appreciably affect transformation efficiency. We find that the transformation efficiency for A. baylyi is a resilient characteristic that remains high under most conditions tested. We do find several distinct conditions that alter natural transformation efficiency including addition of succinate, Fe2+ (ferrous) iron chelation, and substitution of sodium ions with potassium ones. These distinct conditions could be useful to fine tune transformation efficiency for researchers using A. baylyi as a model organism to study natural transformation.

Suppressed translation and ULK1 degradation as potential mechanisms of autophagy limitation under prolonged starvation.

Macroautophagy/autophagy is a well-organized process of intracellular degradation, which is rapidly activated under starvation conditions. Recent data demonstrate a transcriptional upregulation of several autophagy genes as a mechanism that controls autophagy in response to starvation. Here we report that despite the significant upregulation of mRNA of the essential autophagy initiation gene ULK1, its protein level is rapidly reduced under starvation. Although both autophagic and proteasomal systems contribute to the degradation of ULK1, under prolonged nitrogen deprivation, its level was still reduced in ATG7 knockout cells, and only initially stabilized in cells treated with the lysosomal or proteasomal inhibitors. We demonstrate that under starvation, protein translation is rapidly diminished and, similar to treatments with the proteosynthesis inhibitors cycloheximide or anisomycin, is associated with a significant reduction of ULK1. Furthermore, it was found that inhibition of the mitochondrial respiratory complexes or the mitochondrial ATP synthase function that could also take place in the absence of substrates, promote upregulation of ULK1 mRNA and protein expression in an AMPK-dependent manner in U1810 lung cancer cells growing in complete culture medium. These inhibitors could also drastically increase the ULK1 protein in U1810 cells with knockout of ATG13, where the ULK1 expression is significantly diminished. However, such upregulation of ULK1 protein is negligible under starvation conditions, further signifying the contribution of translation and suggesting that transcriptional upregulation of ULK1 protein will be diminished under such conditions. Thus, we propose a model where inhibition of protein translation, together with the degradation systems, limit autophagy during starvation.

Informal caregivers of clients with neurological conditions: profiles, patterns and risk factors for distress from a home care prevalence study.

BACKGROUND: Individuals living in the community with neurological conditions receive the majority of their care from informal caregivers. The purpose of this project was to provide a profile of caregivers of home care clients with neurological conditions. The study also examined prevalence of caregiver distress and the association between neurological conditions and other client and caregiver characteristics with distress. METHODS: The study population included Canadian home care clients in the Winnipeg Regional Health Authority in Manitoba and in the province of Ontario. Clients with RAI-Home Care (RAI-HC) assessment data from 2003 to 2010 were examined. Neurological conditions of interest included Alzheimer's disease and related dementias, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease, epilepsy, muscular dystrophy, cerebral palsy, traumatic brain injury, spinal cord injury, and stroke. Descriptive statistics were analyzed to describe home care client characteristics and caregiver characteristics for each neurological condition. Logistic regression analysis was used to identify risk factors associated with caregiver distress. RESULTS: A substantial proportion of home care clients were found to have one or more of the neurological conditions (38.8% to 41.9%). Caregiver distress was twice as prevalent among caregivers of clients with neurological conditions (28.0%). The largest associations with caregiver distress were the amount of informal care hours provided in a week and the MAPLe algorithm, an indicator of a client's level of priority for care. After adjustment for client characteristics, Huntington disease was the neurological condition most strongly associated with caregiver distress. However, clients' clinical characteristics and informal care hours had a stronger association with caregiver distress than the presence of a neurological condition. Provision of formal home care services provided a protective effect from caregiver distress. CONCLUSIONS: Neurological conditions are common among home care clients and a significant proportion of informal caregivers providing care to these clients experience distress. The complexity of clients with neurological conditions suggests the need for multicomponent support strategies for informal caregivers.

Community palliative care clinical nurse specialists: a descriptive study of nurse-patient interactions.

BACKGROUND: With an ageing population and changes to the UK process of commissioning health-care services, it is important that the role of the community palliative care clinical nurse specialist (CPC-CNS) is better understood. AIM: This study aimed to describe CPC-CNS activities during interactions with patients. METHODS: Four CPC-CNSs were observed and audio-recorded during interactions with 34 patients. The data was assessed qualitatively using thematic analysis. RESULTS: An enormous breadth of activities were observed, within a framework of assessment, planning, intervention, and evaluation. Cross-cutting themes were real-time decision making, leadership, ability to respond to and coordinate complex and varied situations, and communication techniques. Data saturation was not achieved. CONCLUSION: CPC-CNSs provide multifaceted care, requiring wide-ranging knowledge to enable them to act as liaison points in a complex health service, respond independently to the fluctuating needs of patients, and provide effective advance care planning, particularly to those with advanced disease, multi-morbidity, and frailty.

Attitudes to mental illness in the U.K. military: a comparison with the general population.

OBJECTIVES: To compare attitudes to mental illness in the U.K. military and in the general population in England. METHODS: Using data from a cross-sectional survey of 821 U.K. military personnel and a separate cross-sectional survey of 1,729 members of the general population in England, levels of agreement with five statements about mental illness were compared in the military and the general population. RESULTS: The majority of respondents from both populations showed positive attitudes toward mental illness. The general population showed slightly more positive attitudes toward integrating people with mental illness into the community (68.0% [65.7%-70.1%] agreed that "People with mental illness have the same rights to a job as everyone else," vs. 56.7% [51.5%-61.7%] of the military). However, the general population showed more negative attitudes about the causes of mental illness (62.4% [60.1%-64.6%] disagreed that "One of the main causes of mental illness is a lack of self-discipline and willpower," vs. 81.3% [77.0%-84.9%] of the military). CONCLUSIONS: Overall, attitudes toward mental illness are comparable in the general population in England and the U.K. military. Differences included the military holding more positive attitudes about the causes of mental illness, but more negatives attitudes about job rights of those with mental illness. Strategies aiming to improve attitudes toward mental illness could focus particularly on personnel's concerns around mental illness impacting on their career.

Suppressors of cytokine signaling 2 and 3 diametrically control macrophage polarization.

Suppressors of cytokine signaling (SOCS) are important regulators of lipopolysaccharide (LPS) and cytokine responses but their role in macrophage polarization is unknown. We have shown here that myeloid-restricted Socs3 deletion (Socs3(Lyz2cre)) resulted in resistance to LPS-induced endotoxic shock, whereas Socs2(-/-) mice were highly susceptible. We observed striking bias toward M2-like macrophages in Socs3(Lyz2cre) mice, whereas the M1-like population was enriched in Socs2(-/-) mice. Adoptive transfer experiments showed that responses to endotoxic shock and polymicrobial sepsis were transferable and macrophage dependent. Critically, this dichotomous response was associated with enhanced regulatory T (Treg) cell recruitment by Socs3(Lyz2cre) cells, whereas Treg cell recruitment was absent in the presence of Socs2(-/-) macrophages. In addition, altered polarization coincided with enhanced interferon-gamma (IFN-γ)-induced signal transducer and activator of transcription-1 (STAT1) activation in Socs2(-/-) macrophages and enhanced interleukin-4 (IL-4) plus IL-13-induced STAT6 phosphorylation in Socs3(Lyz2cre) macrophages. SOCS, therefore, are essential controllers of macrophage polarization, regulating inflammatory responses.

Siglec-E is up-regulated and phosphorylated following lipopolysaccharide stimulation in order to limit TLR-driven cytokine production.

Although production of cytokines by TLR is essential for viral and bacterial clearance, overproduction can be detrimental, thus controlling these responses is essential. CD33-related sialic acid binding Ig-like lectin receptors (Siglecs) have been implicated in the control of leukocyte responses. In this study, we report that murine Siglec-E is induced by TLRs in a MyD88-specific manner, is tyrosine phosphorylated following LPS stimulation, and negatively regulates TLR responses. Specifically, we demonstrate the Siglec-E expression inhibits TLR-induced NF-kappaB and more importantly, the induction of the antiviral cytokines IFN-beta and RANTES. Siglec-E mediates its inhibitory effects on TIR domain containing adaptor inducing IFN-beta (TRIF)-dependent cytokine production via recruitment of the tyrosine [corrected] phosphatase SHP2 and subsequent inhibition of TBK1 activity as evidenced by enhanced TBK1 phosphorylation in cells following knockdown of Siglec-E expression. Taken together, our results demonstrate a novel role for Siglec-E in controlling the antiviral response to TLRs and thus helping to maintain a healthy cytokine balance following infection.

CCL11 blocks IL-4 and GM-CSF signaling in hematopoietic cells and hinders dendritic cell differentiation via suppressor of cytokine signaling expression.

The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on inflammatory cytokine signaling has not been explored. In this study, we find that CCL11 induces suppressor of cytokine signaling (SOCS)1 and SOCS3 expression in murine macrophages, human monocytes, and dendritic cells (DCs). We also discover that CCL11 inhibits GM-CSF-mediated STAT5 activation and IL-4-induced STAT6 activation in a range of hematopoietic cells. This blockade of cytokine signaling by CCL11 results in reduced differentiation and endocytic ability of DCs, implicating CCL11-induced SOCS as mediators of chemotactic inflammatory control. These findings demonstrate cross-talk between chemokine and cytokine responses, suggesting that myeloid cells tracking to the inflammatory site do not differentiate in the presence of this chemokine, revealing another role for SOCS in inflammatory regulation.

Respiratory syncytial virus NS1 protein degrades STAT2 by using the Elongin-Cullin E3 ligase.

Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.

SOCS3 targets Siglec 7 for proteasomal degradation and blocks Siglec 7-mediated responses.

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Leveraging of open EMR architecture for clinical trial accrual.

Accrual to clinical trials is a major bottleneck in scientific progress in clinical medicine. Many methods for identifying potential subjects and improving accrual have been pursued; few have succeeded, and none have proven generally reproducible or scalable. We leveraged the open architecture of the core clinical data repository of our electronic medical record system to prototype a solution for this problem in a manner consistent with contemporary regulations and research ethics. We piloted the solution with a local investigator-initiated trial for which candidate identification was expected to be difficult. Key results in the eleven months of experience to date include automated screening of 7,296,708 lab results from 69,288 patients, detection of 1,768 screening tests of interest, identification of 70 potential candidates who met all further automated criteria, and accrual of three candidates to the trial. Hypotheses for this disappointing impact on accrual, and directions for future research, are discussed.