Downstream STING pathways IRF3 and NF-κB differentially regulate CCL22 in response to cytosolic dsDNA.

Double-stranded DNA (dsDNA) in the cytoplasm of eukaryotic cells is abnormal and typically indicates the presence of pathogens or mislocalized self-DNA. Multiple sensors detect cytosolic dsDNA and trigger robust immune responses via activation of type I interferons. Several cancer immunotherapy treatments also activate cytosolic nucleic acid sensing pathways, including oncolytic viruses, nucleic acid-based cancer vaccines, and pharmacological agonists. We report here that cytosolic dsDNA introduced into malignant cells can robustly upregulate expression of CCL22, a chemokine responsible for the recruitment of regulatory T cells (Tregs). Tregs in the tumor microenvironment are thought to repress anti-tumor immune responses and contribute to tumor immune evasion. Surprisingly, we found that CCL22 upregulation by dsDNA was mediated primarily by interferon regulatory factor 3 (IRF3), a key transcription factor that activates type I interferons. This finding was unexpected given previous reports that type I interferon alpha (IFN-α) inhibits CCL22 and that IRF3 is associated with strong anti-tumor immune responses, not Treg recruitment. We also found that CCL22 upregulation by dsDNA occurred concurrently with type I interferon beta (IFN-ß) upregulation. IRF3 is one of two transcription factors downstream of the STimulator of INterferon Genes (STING), a hub adaptor protein through which multiple dsDNA sensors transmit their signals. The other transcription factor downstream of STING, NF-κB, has been reported to regulate CCL22 expression in other contexts, and NF-κB has also been associated with multiple pro-tumor functions, including Treg recruitment. However, we found that NF-κB in the context of activation by cytosolic dsDNA contributed minimally to CCL22 upregulation compared with IRF3. Lastly, we observed that two strains of the same cell line differed profoundly in their capacity to upregulate CCL22 and IFN-ß in response to dsDNA, despite apparent STING activation in both cell lines. This finding suggests that during tumor evolution, cells can acquire, or lose, the ability to upregulate CCL22. This study adds to our understanding of factors that may modulate immune activation in response to cytosolic DNA and has implications for immunotherapy strategies that activate DNA sensing pathways in cancer cells.

Cryptic genetic variation enhances primate L1 retrotransposon survival by enlarging the functional coiled coil sequence space of ORF1p.

Accounting for continual evolution of deleterious L1 retrotransposon families, which can contain hundreds to thousands of members remains a major issue in mammalian biology. L1 activity generated upwards of 40% of some mammalian genomes, including humans where they remain active, causing genetic defects and rearrangements. L1 encodes a coiled coil-containing protein that is essential for retrotransposition, and the emergence of novel primate L1 families has been correlated with episodes of extensive amino acid substitutions in the coiled coil. These results were interpreted as an adaptive response to maintain L1 activity, however its mechanism remained unknown. Although an adventitious mutation can inactivate coiled coil function, its effect could be buffered by epistatic interactions within the coiled coil, made more likely if the family contains a diverse set of coiled coil sequences-collectively referred to as the coiled coil sequence space. Amino acid substitutions that do not affect coiled coil function (i.e., its phenotype) could be "hidden" from (not subject to) purifying selection. The accumulation of such substitutions, often referred to as cryptic genetic variation, has been documented in various proteins. Here we report that this phenomenon was in effect during the latest episode of primate coiled coil evolution, which occurred 30-10 MYA during the emergence of primate L1Pa7-L1Pa3 families. First, we experimentally demonstrated that while coiled coil function (measured by retrotransposition) can be eliminated by single epistatic mutations, it nonetheless can also withstand extensive amino acid substitutions. Second, principal component and cluster analysis showed that the coiled coil sequence space of each of the L1Pa7-3 families was notably increased by the presence of distinct, coexisting coiled coil sequences. Thus, sampling related networks of functional sequences rather than traversing discrete adaptive states characterized the persistence L1 activity during this evolutionary event.

Nursing Students' Perceptions of Just Culture in Nursing Programs: A Multisite Study.

BACKGROUND: While just culture is embraced in the clinical setting, just culture has not been systematically incorporated into nursing education. PURPOSE: The purpose of this study was to assess prelicensure nursing student perceptions of just culture in academia. METHODS: Following a quantitative, descriptive design, the Just Culture Assessment Tool for Nursing Education (JCAT-NE) was used to measure just culture across multiple (N = 15) nursing programs. RESULTS: The majority of JCAT-NE respondents (78%) reported their program has a safety reporting system, 15.4% had involvement in a safety-related event, and 12% submitted an error report. The JCAT-NE mean total score was 127.4 (SD, 23.6), with a statistically significant total score decline as students progressed from the beginning (133.6 [SD, 20.52]) to the middle (129.77 [SD, 23.6]) and end (122.2 [SD, 25.43]) of their programs (χ[2] = 25.09, P < .001). CONCLUSIONS: The results from this study are a call to action for nursing education to emphasize the tenets of just culture, error reporting, and quality improvement.

Transient evolution of flow profiles in a shear banding wormlike micellar solution: experimental results and a comparison with the VCM model.

In this paper we investigate the flow of a shear banding wormlike micellar fluid based on cetyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal). The flow is studied in a custom-built Taylor-Couette (TC) cell via a combination of particle tracking velocimetry and in situ rheology. The spatiotemporal evolution of the velocity profile across the rheometer gap is tracked after an imposed step in the shear rate. In a range of shear rates the mixture shows shear banding behavior, that is distinct and differing shear rate profiles across the gap. As the shear bands form temporally, an elastic recoil including negative velocity (that is in the opposite direction to that of the imposed motion) is observed in a subset of the gap. While elastic recoil has been reported in experiments on monodisperse polymers [S. Ravindranath, et al., Macromolecules, 2008, 41, 2663-2670], on a wormlike micellar solution in a cone-plate rheometer [P. E. Boukany and S. Q. Wang, Macromolecules, 2008, 41(4), 1455-1464], and in theoretical studies [L. Zhou, et al., J. Non-Newtonian Fluid Mech., 2014, 211, 70-83; J. M. Adams, et al., J. Rheol., 2011, 55, 1007-1032] of wormlike micellar flows, it has not been previously reported in experiments on shear banding wormlike micelles in Taylor-Couette flows. Additionally, the mixture shows significant wall slip at the outer (stationary) Couette cylinder at high shear rates. Experimental results are compared to simulations of models of wormlike micelles, particularly the VCM model [L. Zhou, et al., J. Non-Newtonian Fluid Mech., 2014, 211, 70-83]. There are differences between the experimental results for this fluid and those reported previously. The difference arises from the size of the elasticity number which for the fluid reported in the paper is four orders of magnitude larger than that of other preparations.

A Review of Four Health Procedures That School Nurses May Encounter.

The school nurse plays a vital role in providing care and meeting the health needs of students in the school setting. Students attend school with chronic conditions and complex medical problems such as quadriplegia, cerebral palsy, spina bifida, and muscular dystrophy. It is the responsibility of the school nurse to provide appropriate assessment, early intervention, and care for children in the school environment. The purpose of this article is to review and discuss the knowledge and skills the school nurse needs to care for students with central venous lines, gastrostomy tubes, altered urinary elimination, and tracheostomies.

Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.

Immune-profiling is becoming an important tool to identify predictive markers for the response to immunotherapy. Our goal was to validate multiplex immunofluorescence (mIF) panels to apply to formalin-fixed and paraffin-embedded tissues using a set of immune marker antibodies, with the Opal™ 7 color Kit (PerkinElmer) in the same tissue section. We validated and we described two panels aiming to characterize the expression of PD-L1, PD-1, and subsets of tumor associated immune cells. Panel 1 included pancytokeratin (AE1/AE3), PD-L1, CD4, CD8, CD3, CD68, and DAPI, and Panel 2 included pancytokeratin, PD-1, CD45RO, granzyme B, CD57, FOXP3, and DAPI. After all primary antibodies were tested in positive and negative controls by immunohistochemistry and uniplex IF, panels were developed and simultaneous marker expressions were quantified using the Vectra 3.0™ multispectral microscopy and image analysis InForm™ 2.2.1 software (PerkinElmer).These two mIF panels demonstrated specific co-localization in different cells that can identify the expression of PD-L1 in malignant cells and macrophages, and different T-cell subpopulations. This mIF methodology can be an invaluable tool for tumor tissue immune-profiling to allow multiple targets in the same tissue section and we provide that is accurate and reproducible method when is performed carefully under pathologist supervision.

Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition.

BACKGROUND: The Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity. RESULTS: We found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls. CONCLUSIONS: Engineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

The challenge of ORF1p phosphorylation: Effects on L1 activity and its host.

L1 non-LTR retrotransposons are autonomously replicating genetic elements that profoundly affected their mammalian hosts having generated upwards of 40% or more of their genomes. Although deleterious, they remain active in most mammalian species, and thus the nature and consequences of the interaction between L1 and its host remain major issues for mammalian biology. We recently showed that L1 activity requires phosphorylation of one of its 2 encoded proteins, ORF1p, a nucleic acid chaperone and the major component of the L1RNP retrotransposition intermediate. Reversible protein phosphorylation, which is effected by interacting cascades of protein kinases, phosphatases, and ancillary proteins, is a mainstay in the regulation and coordination of many basic biological processes. Therefore, demonstrating phosphorylation-dependence of L1 activity substantially enlarged our knowledge of the scope of L1 / host interaction. However, developing a mechanistic understanding of what this means for L1 or its host is a formidable challenge, which we discuss here.

L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling.

Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution.

Phosphorylation of ORF1p is required for L1 retrotransposition.

Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.

HTLV-1 HBZ protein deregulates interactions between cellular factors and the KIX domain of p300/CBP.

The complex retrovirus human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. Deregulation of cellular transcription is thought to be an important step for T-cell transformation caused by viral infection. HTLV-1 basic leucine zipper factor (HBZ) is one of the viral proteins believed to be involved in this process, as it deregulates the expression of numerous cellular genes. In the context of the provirus, HBZ represses HTLV-1 transcription, in part, by binding to the homologous cellular coactivators p300 and CBP. These coactivators play a central role in transcriptional regulation. In this study, we determined that HBZ binds with high affinity to the KIX domain of p300/CBP. This domain contains two binding surfaces that are differentially targeted by multiple cellular factors. We show that two φXXφφ motifs in the activation domain of HBZ mediate binding to a single surface of the KIX domain, the mixed-lineage leukemia (MLL) binding surface. Formation of this interaction inhibits binding of MLL to the KIX domain while enhancing the binding of the transcription factor c-Myb to the opposite surface of KIX. Consequently, HBZ inhibits transcriptional activation mediated by MLL and enhances activation mediated by c-Myb. CREB, which binds the same surface of KIX as c-Myb, also exhibited an increase in activity through HBZ. These results indicate that HBZ is able to alter gene expression by competing with transcription factors for the occupancy of one surface of KIX while enhancing the binding of factors to the other surface.

Dynamics of raltegravir resistance profile in an HIV type 2-infected patient.

The evolutionary dynamics of RAL resistance in the HIV-2 virus were examined through population and clonal sequence analysis of the IN from baseline, during treatment, and after stopping RAL therapy. The treatment failure of an RAL regimen in the HIV-2 patient studied was associated with the emergence of mutations via the N155H resistance pathway and subsequent switching to the Y143C mutational route. This study has also identified four novel secondary mutations, Q91R, S147G, A153G, and M183I, not previously reported in HIV-1 patients failing RAL therapy. Resistant variants involving the Y143C pathway were noted to have persisted beyond 4 weeks following the cessation of RAL therapy. All resistance-associated mutations were lost at 20 weeks after stopping RAL therapy. Our findings provide evidence supporting the supposition that substantial cross-resistance between strand transfer IN-Is is likely in HIV-2 as shown in HIV-1.

The dynamics of appearance and disappearance of HIV-1 integrase mutations during and after withdrawal of raltegravir therapy.

OBJECTIVES: To monitor HIV-1 integrase resistance mutations during raltegravir (RAL) therapy, including the impact of RAL interruption. DESIGN AND METHOD: An analysis of viral load and the HIV-1 integrase gene evolution in 26 HIV-1 treatment-experienced patients undergoing RAL therapy. RESULTS: Initial suppression of viral load was observed in all patients; however, four patients failed to maintain suppression and subsequently developed resistance at viral load rebound. Mutations Q148R (2 months) followed by G140A/Q148R and then G140A/Y143CHR/Q148R/G163R were detected in the virus from one patient, and these reverted to wild type when treatment was withdrawn, although clonal analysis identified maintenance of RAL resistance minority species at this time point. RAL treatment was restarted after 6 months, and 2 weeks later, Y143CY/G163RG mutations appeared. In three other patients, viruses with N155H emerged at viral rebound either alone (2 months), followed by V151I (8 months) or alone (10 months), or together with V151I/G163RG (7 months). Loss of virus with the N155H mutation occurred in these patients when RAL therapy was terminated, despite maintenance of reverse transcriptase/polymerase resistance mutations. CONCLUSION: Complete viral suppression was important in order to prevent resistance emerging. RAL-resistance mutations were detected in the presence of other antiviral treatments, and the reverse of these mutations following RAL cessation suggests that a fitness deficit was conferred by these mutants. The observation that following RAL interruption virus rebound was with previously existing reverse transcriptase/polymerase mutations in the absence of integrase mutations implies that it is pre-RAL-archived viruses that re-emerge.

An interaction between the human T cell leukemia virus type 1 basic leucine zipper factor (HBZ) and the KIX domain of p300/CBP contributes to the down-regulation of tax-dependent viral transcription by HBZ.

Activation of human T cell leukemia virus type 1 (HTLV-1) transcription is established through the formation of protein complexes on the viral promoter that are essentially composed of the cellular basic leucine zipper (bZIP) transcription factor cAMP-response element-binding protein (CREB (or certain other members of the ATF/CREB family), the HTLV-1-encoded transactivator Tax, and the pleiotropic cellular coactivators p300/CBP. HTLV-1 bZIP factor (HBZ) is a protein encoded by HTLV-1 that contains a bZIP domain and functions to repress HTLV-1 transcription. HBZ has been shown to repress viral transcription by dimerizing with CREB, which occurs specifically through the bZIP domain in each protein, and preventing CREB from binding to the DNA. However, we previously found that HBZ causes only partial removal of CREB from a chromosomally integrated viral promoter, and more importantly, an HBZ mutant lacking the COOH-terminal bZIP domain retains the ability to repress viral transcription. These results suggest that an additional mechanism contributes to HBZ-mediated repression of HTLV-1 transcription. In this study, we show that HBZ binds directly to the p300 and CBP coactivators. Two LXXLL-like motifs located within the NH(2)-terminal region of HBZ are important for this interaction and specifically mediate binding to the KIX domain of p300/CBP. We provide evidence that this interaction interferes with the ability of Tax to bind p300/CBP and thereby inhibits the association of the coactivators with the viral promoter. Our findings demonstrate that HBZ utilizes a bipartite mechanism to repress viral transcription.

Salmonella-induced SipB-independent cell death requires Toll-like receptor-4 signalling via the adapter proteins Tram and Trif.

Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen that causes macrophage cell death by at least two different mechanisms. Rapid cell death is dependent on the Salmonella pathogenicity island-1 protein SipB whereas delayed cell death is independent of SipB and occurs 18-24 hr post infection. Lipopolysaccharide (LPS) is essential for the delayed cell death. LPS is the main structural component of the outer membrane of Gram-negative bacteria and is recognized by Toll-like receptor 4, signalling via the adapter proteins Mal, MyD88, Tram and Trif. Here we show that S. typhimurium induces SipB-independent cell death through Toll-like receptor 4 signalling via the adapter proteins Tram and Trif. In contrast to wild type bone marrow derived macrophages (BMDM), Tram(-/-) and Trif(-/-) BMDM proliferate in response to Salmonella infection.

Retention of African-American students in baccalaureate nursing programs: are we doing enough?

With the increasing minority population in the United States, much attention has been given to the lack of diversity among health care professionals, specifically nursing. Since the 1960s, the federal government has provided financial resources to institutions of higher education whose purpose was to diversify the health care profession. Historically, these resources have supported initiatives that primarily focused on the recruitment of minority students into higher education. These efforts temporarily increased the enrollment of students from varying racial and ethnic backgrounds. However, without established retention initiatives in place, the attrition rates for students from diverse backgrounds far exceeded the enrollment rates. Consequently, the nursing workforce continues to be a predominantly White female profession. In order for schools of nursing to create a workforce reflective of its patient population, both nursing education and institutions of higher education must be committed to implementing initiatives to increase the retention and graduation rates of minority students.

Development of an anti-core lipopolysaccharide vaccine for the prevention and treatment of sepsis.

Sepsis continues to be a leading cause of death among hospitalized patients. Despite advances in supportive care and the availability of potent antimicrobials, the mortality exceeds 20%. The passive infusion of antibodies directed against a conserved region of the lipopolysaccharide (LPS) of Gram-negative bacteria was highly protective in an early study (NEJM 307 [1982] 1225). When this and similar preparations were unable to show consistent efficacy, efforts were directed towards other strategies, including cytokine modulation. Our group found that a whole bacterial vaccine made from the Escherichia coli O111:B4, J5 (Rc chemotype) mutant induced protective antibodies when given passively as treatment for sepsis in a neutropenic rat model. A subunit vaccine, composed of detoxified J5 LPS complexed to group B meningococcal outer membrane protein (OMP), provided similar protection when antibodies were given passively, or induced actively in both the neutropenic and cecal ligation/puncture models of sepsis. A phase I study in 24 subjects (at 5, 10 and 25 microg doses [based on LPS] for each group of 8) revealed the vaccine to be well-tolerated with no systemic endotoxin-like effects. Although a two to three-fold increase in antibody levels over baseline (by ELISA assay) was observed at the 10 and 25 microg doses, the plasma from both high and low responders reduced LPS-induced cytokine generation in whole blood. Reimmunization of six subjects at 12 months did not convert low responders to high responders or boost the still elevated anti-J5 LPS levels of high responders. If functional assays of anti-LPS antibodies are better predictors of vaccine efficacy than ELISA antibody levels, then it will be necessary to determine which of many potential assays best correlates with protection in animal models. We are currently comparing a panel of functional assays with protective efficacy in animal models of sepsis, as well as the ability of adjuvants to enhance vaccine efficacy. The availability of an effective anti-endotoxin vaccine will provide additional therapeutic options for the prevention and/or treatment of sepsis.

Phase I study of detoxified Escherichia coli J5 lipopolysaccharide (J5dLPS)/group B meningococcal outer membrane protein (OMP) complex vaccine in human subjects.

We previously observed that a detoxified Escherichia coli O111, Rc chemotype J5 lipopolysaccharide (J5dLPS)/group B meningococcal outer membrane protein (OMP) vaccine protected animals from experimental lethal sepsis when immune antibodies were given passively as treatment at the onset of fever or when vaccine was given actively as prophylaxis. To test the safety and immunogenicity of this vaccine, we administered doses of 5, 10 and 25 microg (based on dLPS) of vaccine at days 0, 28 and 56 to 24 human subjects (8 per group). Temperatures of 100.3, 99.5 and 99.4 degrees F occurred in three subjects. At 24h, pain at the injection site was moderate in 38%, mild in 44% and not present in 18%, while at 48 h, it was 1, 25 and 73%, respectively. No alterations in baseline renal, hepatic or hematologic functions occurred. There were two to three times mean-fold increases in anti-J5dLPS IgG (range: 1.9-5.1) and IgM (range: 1.2-9.2) levels in subjects receiving the 10 and 25 microg doses. At 12-month follow-up, three of the original responders had continued elevation of antibody levels. A 25 microg booster dose of vaccine did not increase antibody levels among those responders and did not elicit antibodies among three subjects with no previous antibody response. The plasma from the six volunteers inhibited LPS-induced cytokine generation in human whole blood ex vivo. We conclude that this J5dLPS/OMP vaccine was safe and well-tolerated with transient, local pain at the injection site. Vaccine formulations with different adjuvants are currently under investigation.