Rapamycin-induced inhibition of HTLV-I LTR activity is rescued by c-Myb.

BACKGROUND: Rapamycin is an immunosuppressive which represses translation of transcripts harbouring a polypyrimidine motif downstream of the mRNA cap site through the mammalian target of rapamycin complex. It inhibits the abnormal autologous proliferation of T-cell clones containing a transcriptionally active human T-lymphotropic virus, type I (HTLV-I) provirus, generated from infected subjects. We showed previously that this effect is independent of the polypyrimidine motifs in the viral long terminal repeat (LTR) R region suggesting that HTLV-I transcription, and not translation, is being affected. Here we studied whether rapamycin is having an effect on a specific transcription factor pathway. Further, we investigated whether mRNAs encoding transcription factors involved in HTLV-I transcriptional activation, specifically CREB, Ets and c-Myb, are implicated in the rapamycin-sensitivity of the HTLV-I LTR. RESULTS: An in vitro analysis of the role of SRE- and NF-kappaB-mediated transcription highlighted the latter as rapamycin sensitive. Over-expression of c-Myb reversed the rapamycin effect. CONCLUSION: The sensitivity of HTLV-I transcription to rapamycin may be effected through an NF-kappaB-pathway associated with the rapamycin-sensitive mTORC1 cellular signalling network.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Debating standard language ideology in the classroom:Using the speak good English movement to raise awareness of Global Englishes

In this article, we describe and evaluate an innovative pedagogical task designed to raise awareness of Global Englishes and to challenge standard language ideology in an English language classroom. The task encouraged the learning and debate of the controversial Speak Good English Movement, which campaigns for Singaporeans to use a ‘standard’ form of English rather than the local variety, ‘Singlish’. The debate was introduced as a Global Englishes-inspired multi-lesson task at a Japanese university with 108 students in four classes. Data were collected in the form of written reflections, in which learners stated their own positions and beliefs. Data indicated that the majority sided against the Speak Good English Movement, viewing Singlish as a legitimate variety of English with important cultural attributes, which contributed to a national identity. Results showed that the debate achieved the aim of encouraging students to critically reflect on standard language ideology in both English and Japanese, their mother tongue. Overall, the activity was successful in raising awareness of the diversity of English, and encouraging critical reflection on the complexities surrounding standard language beliefs. This was seen to be essential given the dominance of the ‘native’ speaker episteme in Japanese language curricula

Genetic diversity in the env V1-V2 region of proviral quasispecies from long-term controller MHC-typed cynomolgus macaques infected with SHIVSF162P4cy

Intra-host evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) has been shown by viral RNA analysis in subjects who naturally suppress plasma viremia to low levels, known as controllers. However, little is known about the variability of proviral DNA and the inter-relationships among contained systemic viremia, rate of reservoir reseeding and specific major histocompatibility complex (MHC) genotypes, in controllers. Here, we analysed the proviral DNA quasispecies of the env V1-V2 region, in PBMCs and in anatomical compartments of 13 long-term controller monkeys after 3.2 years of infection with simian/human immunodeficiency virus (SHIV)SF162P4cy. A considerable variation in the genetic diversity of proviral quasispecies was present among animals. Seven monkeys exhibited env V1-V2 proviral populations composed of both clusters of identical ancestral sequences and new variants, whereas the other six monkeys displayed relatively high env V1-V2 genetic diversity with a large proportion of diverse novel sequences. Our results demonstrate that in SHIVSF162P4cy-infected monkeys there exists a disparate pattern of intra-host viral diversity and that reseeding of the proviral reservoir occurs in some animals. Moreover, even though no particular association has been observed between MHC haplotypes and the long-term control of infection, a remarkably similar pattern of intra-host viral diversity and divergence was found within animals carrying the M3 haplotype. This suggests that in animals bearing the same MHC haplotype and infected with the same virus, viral diversity follows a similar pattern with similar outcomes and control of infection

GB Virus B and Hepatitis C Virus, Distantly Related Hepaciviruses, Share an Entry Factor, Claudin-1

Due to increased and broadened screening efforts, the last decade has seen a rapid expansion in the number of viral species classified into the Hepacivirus genus. Conserved genetic features of hepaciviruses suggest that they have undergone specific adaptation and have evolved to hijack similar host proteins for efficient propagation in the liver. Here, we developed pseudotyped viruses to elucidate the entry factors of GB virus B (GBV-B), the first hepacivirus described in an animal after hepatitis C virus (HCV). GBV-B-pseudotyped viral particles (GBVBpp) were shown to be uniquely sensitive to the sera of tamarins infected with GBV-B, validating their usefulness as a surrogate for GBV-B entry studies. We screened GBVBpp infection of human hepatoma cell lines that were CRISPR/Cas9 engineered to ablate the expression of individual HCV receptors/entry factors and found that claudin-1 is essential for GBV-B infection, indicating the GBV-B and HCV share an entry factor. Our data suggest that claudin-1 facilitates HCV and GBV-B entry through distinct mechanisms since the former requires the first extracellular loop and the latter is reliant on a C-terminal region containing the second extracellular loop. The observation that claudin-1 is an entry factor shared between these two hepaciviruses suggests that the tight junction protein is of fundamental mechanistic importance during cell entry. IMPORTANCE Hepatitis C virus (HCV) is a major public health burden; approximately 58 million individuals have chronic HCV infection and are at risk of developing cirrhosis and liver cancer. To achieve the World Health Organization's target of eliminating hepatitis by 2030, new therapeutics and vaccines are needed. Understanding how HCV enters cells can inform the design of new vaccines and treatments targeting the first stage of infection. However, the HCV cell entry mechanism is complex and has been sparsely described. Studying the entry of related hepaciviruses will increase the knowledge of the molecular mechanisms of the first stages of HCV infection, such as membrane fusion, and inform structure-guided HCV vaccine design; in this work, we have identified a protein, claudin-1, that facilitates the entry of an HCV-related hepacivirus but with a mechanism not described for HCV. Similar work on other hepaciviruses may unveil a commonality of entry factors and, possibly, new mechanisms

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

Background Adoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long term protection due to T cell exhaustion induced by chronic TCR stimulation in the tumor microenvironment. One benefit of ACT, however, is that it allows for cellular manipulations, such as deletion of the phosphotyrosine phosphatase non-receptor type 22 (PTPN22), which improves CD8+ T cell anti-tumor efficacy in ACT. We tested whether Ptpn22KO cytolytic T cells (CTL) were also more effective than Ptpn22WT CTL in controlling tumors in scenarios that favor T cell exhaustion. Methods Tumor control by Ptpn22WT and Ptpn22KO CTL was assessed following adoptive transfer of low numbers of CTL to mice with subcutaneously implanted MC38 tumors. Tumor infiltrating lymphocytes were isolated for analysis of effector functions. An in vitro assay was established to compare CTL function in response to acute and chronic re-stimulation with antigen-pulsed tumor cells. The expression of effector and exhaustion-associated proteins by Ptpn22WT and Ptpn22KO T cells was followed over time in vitro and in vivo using the ID8 tumor model. Finally, the effect of PD-1 and TIM-3 blockade on Ptpn22KO CTL tumor control was assessed using monoclonal antibodies and CRISPR/Cas9-mediated knockout. Results Despite having improved effector function at the time of transfer, Ptpn22KO CTL became more exhausted than Ptpn22WT CTL, characterized by more rapid loss of effector functions, and earlier and higher expression of inhibitory receptors (IRs), particularly the terminal exhaustion marker TIM-3. TIM-3 expression, under the control of the transcription factor NFIL3, was induced by IL-2 signaling which was enhanced in Ptpn22KO cells. Anti-tumor responses of Ptpn22KO CTL were improved following PD-1 blockade in vivo, yet knockout or antibody-mediated blockade of TIM-3 did not improve but further impaired tumor control, indicating TIM-3 signaling itself did not drive the diminished function seen in Ptpn22KO CTL. Conclusions This study questions whether TIM-3 plays a role as an IR and highlights that genetic manipulation of T cells for ACT needs to balance short term augmented effector function against the risk of T cell exhaustion in order to achieve longer term protection. What is already known on this topic • T cell exhaustion in the tumor microenvironment is a major factor limiting the potential success of adoptive cell therapy (ACT) in the treatment of solid tumors. • Deletion of the phosphatase PTPN22 in CD8+ T cells improves their response to tumors, but it is not known whether this influences development of exhaustion. What this study adds • Under conditions which promote exhaustion, CTL lacking PTPN22 exhaust more rapidly than WT cells, despite displaying enhanced effector function in their initial response to antigen. • Ptpn22KO CTL express high levels of the inhibitory receptor TIM-3, but TIM-3 signaling does not directly contribute to Ptpn22KO CTL dysfunction. • Ptpn22KO T cells are more responsive to IL-2 through JAK-STAT signaling, which induces TIM-3 expression via the transcription factor NFIL3. How this study might affect research, practice or policy • Strategies aimed at augmenting T cell effector function for ACT should balance improved responses against an increased risk of T cell exhaustion

‘Talent-spotting’ or ‘social magic’? Inequality, cultural sorting and constructions of the ideal graduate in elite professions

Graduate outcomes – including rates of employment and earnings – are marked by persistent inequalities related to social class, as well as gender, ethnicity and institution. Despite national policy agendas related to social mobility and ‘fair access to the professions’, high-status occupations are disproportionately composed of those from socially privileged backgrounds, and evidence suggests that in recent decades many professions have become less socially representative. This article makes an original contribution to sociological studies of inequalities in graduate transitions and elite reproduction through a distinct focus on the ‘pre-hiring’ practices of graduate employers. It does this through a critical analysis of the graduate recruitment material of two popular graduate employers. It shows how, despite espousing commitments to diversity and inclusion, constructions of the ‘ideal’ graduate privilege individuals who can mobilise and embody certain valued capitals. Using Bourdieusian concepts of ‘social magic’ and ‘institutional habitus’, the article argues that more attention must be paid to how graduate employers’ practices constitute tacit processes of social exclusion and thus militate against the achievement of more equitable graduate outcomes and fair access to the ‘top jobs

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus)

AbstractFlaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3+ lymphocytes decreased and CD20+ lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4+ T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection

Reflections and Experiences of a Co-Researcher involved in a Renal Research Study

Background Patient and Public Involvement (PPI) is seen as a prerequisite for health research. However, current Patient and public involvement literature has noted a paucity of recording of patient and public involvement within research studies. There have been calls for more recordings and reflections, specifically on impact. Renal medicine has also had similar criticisms and any reflections on patient and public involvement has usually been from the viewpoint of the researcher. Roles of patient and public involvement can vary greatly from sitting on an Advisory Group to analysing data. Different PPI roles have been described within studies; one being a co-researcher. However, the role of the co-researcher is largely undefined and appears to vary from study to study. Methods The aims of this paper are to share one first time co-researcher's reflections on the impact of PPI within a mixed methods (non-clinical trial) renal research study. A retrospective, reflective approach was taken using data available to the co-researcher as part of the day-to-day research activity. Electronic correspondence and documents such as meeting notes, minutes, interview thematic analysis and comments on documents were re-examined. The co-researcher led on writing this paper. Results This paper offers a broad definition of the role of the co-researcher. The co-researcher reflects on undertaking and leading on the thematic analysis of interview transcripts, something she had not previously done before. The co-researcher identified a number of key themes; the differences in time and responsibility between being a coresearcher and an Advisory Group member; how the role evolved and involvement activities could match the co-researchers strengths (and the need for flexibility); the need for training and support and lastly, the time commitment. It was also noted that it is preferable that a co-researcher needs to be involved from the very beginning of the grant application. Conclusions The reflections, voices and views of those undertaking PPI has been largely underrepresented in the literature. The role of co-researcher was seen to be rewarding but demanding, requiring a large time commitment. It is hoped that the learning from sharing this experience will encourage others to undertake this role, and encourage researchers to reflect on the needs of those involved.Peer reviewedFinal Published versio

The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the ApcMin/+ mouse small intestine.

BACKGROUND: Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmDelta4A/tmDelta4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively. METHODS: The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's t-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-ras activating mutation. RESULTS: The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology. CONCLUSION: The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are