Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

ATRX modulates the escape from a telomere crisis.

Telomerase activity is the principal telomere maintenance mechanism in human cancers, however 15% of cancers utilise a recombination-based mechanism referred to as alternative lengthening of telomeres (ALT) that leads to long and heterogenous telomere length distributions. Loss-of-function mutations in the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) gene are frequently found in ALT cancers. Here, we demonstrate that the loss of ATRX, coupled with telomere dysfunction during crisis, is sufficient to initiate activation of the ALT pathway and that it confers replicative immortality in human fibroblasts. Additionally, loss of ATRX combined with a telomere-driven crisis in HCT116 epithelial cancer cells led to the initiation of an ALT-like pathway. In these cells, a rapid and precise telomeric elongation and the induction of C-circles was observed; however, this process was transient and the telomeres ultimately continued to erode such that the cells either died or the escape from crisis was associated with telomerase activation. In both of these instances, telomere sequencing revealed that all alleles, irrespective of whether they were elongated, were enriched in variant repeat types, that appeared to be cell-line specific. Thus, our data show that the loss of ATRX combined with telomere dysfunction during crisis induces the ALT pathway in fibroblasts and enables a transient activation of ALT in epithelial cells.

Oral care, loss of personal identity and dignity in residential care homes.

OBJECTIVE: The UK National Institute of Clinical Excellence (NICE) guidelines recommend that care staff who provide daily personal care to residents: "Understand the importance of residents' oral health and the potential effect on their general health, well-being and dignity." The aim of this study was to explore residents' views and perspectives of dental care in care homes in order to understand how to deliver this care. METHOD: Care homes were identified using care home inspection reports for Wales, the UK. Care homes for older people with residents having mental capacity to consent were invited to participate. Data were collected using semi-structured one-to-one interviews with care home residents, care home managers and oral healthcare leads. Interviews were audio recorded, transcribed and analysed using a thematic approach to data. Analysis was assisted by NVivo 10 software. Data collection was completed when no new themes emerged. RESULTS: This analysis presents findings from 26 interviews with residents, across five care homes. Going into care was associated with a loss of identity. Having teeth and looking after teeth (natural teeth or dentures) was part of keeping that identity. All prioritised privacy, pride and independence above effective oral hygiene. Oral hygiene was viewed as a very private event. Carers adapted oral care, to balance time constraints, care, privacy and dignity. Teeth were a part of personal pride to the extent that two residents said they did not want to die without dentures in their mouths. CONCLUSION: Whilst oral care is important to residents, dignity and privacy are often more important; care routines and practices are adapted around this. Carers need to adopt an individualised, pragmatic and sensitive approach to oral care to account for personal dignity when looking after residents to be able to provide appropriate oral care in accordance with guidance. Members of the dental team need to support carers to provide effective oral care, which allows dignified and effective care.

Chromothripsis during telomere crisis is independent of NHEJ, and consistent with a replicative origin.

Telomere erosion, dysfunction, and fusion can lead to a state of cellular crisis characterized by large-scale genome instability. We investigated the impact of a telomere-driven crisis on the structural integrity of the genome by undertaking whole-genome sequence analyses of clonal populations of cells that had escaped crisis. Quantification of large-scale structural variants revealed patterns of rearrangement consistent with chromothripsis but formed in the absence of functional nonhomologous end-joining pathways. Rearrangements frequently consisted of short fragments with complex mutational patterns, with a repair topology that deviated from randomness showing preferential repair to local regions or exchange between specific loci. We find evidence of telomere involvement with an enrichment of fold-back inversions demarcating clusters of rearrangements. Our data suggest that chromothriptic rearrangements caused by a telomere crisis arise via a replicative repair process involving template switching.

Integrative analysis of spontaneous CLL regression highlights genetic and microenvironmental interdependency in CLL.

Spontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.

If the public can vaccinate, why not students? Review of a student nurse placement in a mass vaccination centre.

Delivery of the COVID-19 vaccine has been made possible in part through the use of mass vaccination centres (MVCs). The primary legal framework underpinning the MVC programme is a national protocol enabling registered and non-registered healthcare workers to contribute to the safe and effective administration of the vaccine. The national protocol provided a vehicle for an innovative supervised student nurse placement within an MVC in south Wales. This placement, for undergraduate pre-registration student nurses, formed part of a service improvement project. Through student feedback prior to, and following, the short placement, the learning was unequivocal in terms of knowledge and skills acquisition related to safe and effective vaccine administration, with students providing clear feedback on the positive nature of the placement experience. A placement within an MVC offers a rich educational experience for student nurses, which as yet appears to be underutilised across the UK.

Human TSCM cell dynamics in vivo are compatible with long-lived immunological memory and stemness.

Adaptive immunity relies on the generation and maintenance of memory T cells to provide protection against repeated antigen exposure. It has been hypothesised that a self-renewing population of T cells, named stem cell-like memory T (TSCM) cells, are responsible for maintaining memory. However, it is not clear if the dynamics of TSCM cells in vivo are compatible with this hypothesis. To address this issue, we investigated the dynamics of TSCM cells under physiological conditions in humans in vivo using a multidisciplinary approach that combines mathematical modelling, stable isotope labelling, telomere length analysis, and cross-sectional data from vaccine recipients. We show that, unexpectedly, the average longevity of a TSCM clone is very short (half-life < 1 year, degree of self-renewal = 430 days): far too short to constitute a stem cell population. However, we also find that the TSCM population is comprised of at least 2 kinetically distinct subpopulations that turn over at different rates. Whilst one subpopulation is rapidly replaced (half-life = 5 months) and explains the rapid average turnover of the bulk TSCM population, the half-life of the other TSCM subpopulation is approximately 9 years, consistent with the longevity of the recall response. We also show that this latter population exhibited a high degree of self-renewal, with a cell residing without dying or differentiating for 15% of our lifetime. Finally, although small, the population was not subject to excessive stochasticity. We conclude that the majority of TSCM cells are not stem cell-like but that there is a subpopulation of TSCM cells whose dynamics are compatible with their putative role in the maintenance of T cell memory.

Symptoms of Prenatal Depression Associated with Shorter Telomeres in Female Placenta.

BACKGROUND: Depression is a common mood disorder during pregnancy impacting one in every seven women. Children exposed to prenatal depression are more likely to be born at a low birth weight and develop chronic diseases later in life. A proposed hypothesis for this relationship between early exposure to adversity and poor outcomes is accelerated aging. Telomere length has been used as a biomarker of cellular aging. We used high-resolution telomere length analysis to examine the relationship between placental telomere length distributions and maternal mood symptoms in pregnancy. METHODS: This study utilised samples from the longitudinal Grown in Wales (GiW) study. Women participating in this study were recruited at their presurgical appointment prior to a term elective caesarean section (ELCS). Women completed the Edinburgh Postnatal Depression Scale (EPDS) and trait subscale of the State-Trait Anxiety Inventory (STAI). Telomere length distributions were generated using single telomere length analysis (STELA) in 109 term placenta (37-42 weeks). Multiple linear regression was performed to examine the relationship between maternally reported symptoms of depression and anxiety at term and mean placental telomere length. RESULTS: Prenatal depression symptoms were significantly negatively associated with XpYp telomere length in female placenta (B = -0.098, p = 0.026, 95% CI -0.184, -0.012). There was no association between maternal depression symptoms and telomere length in male placenta (B = 0.022, p = 0.586, 95% CI -0.059, 0.103). There was no association with anxiety symptoms and telomere length for either sex. CONCLUSION: Maternal prenatal depression is associated with sex-specific differences in term placental telomeres. Telomere shortening in female placenta may indicate accelerated placental aging.

Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells.

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as "anticancer compounds" and "anticancer epi-compounds" and PK083 a "damage-corrective" compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

CCR8 Expression Defines Tissue-Resident Memory T Cells in Human Skin.

Human skin harbors two major T cell compartments of equal size that are distinguished by expression of the chemokine receptor CCR8. In vitro studies have demonstrated that CCR8 expression is regulated by TCR engagement and the skin tissue microenvironment. To extend these observations, we examined the relationship between CCR8+ and CCR8- skin T cells in vivo. Phenotypic, functional, and transcriptomic analyses revealed that CCR8+ skin T cells bear all the hallmarks of resident memory T cells, including homeostatic proliferation in response to IL-7 and IL-15, surface expression of tissue localization (CD103) and retention (CD69) markers, low levels of inhibitory receptors (programmed cell death protein 1, Tim-3, LAG-3), and a lack of senescence markers (CD57, killer cell lectin-like receptor subfamily G member 1). In contrast, CCR8- skin T cells are heterogeneous and comprise variable numbers of exhausted (programmed cell death protein 1+), senescent (CD57+, killer cell lectin-like receptor subfamily G member 1+), and effector (T-bethi, Eomeshi) T cells. Importantly, conventional and high-throughput sequencing of expressed TCR ß-chain (TRB) gene rearrangements showed that these CCR8-defined populations are clonotypically distinct, suggesting unique ontogenies in response to separate antigenic challenges and/or stimulatory conditions. Moreover, CCR8+ and CCR8- skin T cells were phenotypically stable in vitro and displayed similar levels of telomere erosion, further supporting the likelihood of a nonlinear differentiation pathway. On the basis of these results, we propose that long-lived memory T cells in human skin can be defined by the expression of CCR8.

Human Endometrial Carcinogenesis Is Associated with Significant Reduction in Long Non-Coding RNA, TERRA.

Telomeres are transcribed as long non-coding RNAs called TERRAs (Telomeric repeat containing RNA) that participate in a variety of cellular regulatory functions. High telomerase activity (TA) is associated with endometrial cancer (EC). This study aimed to examine the levels of three TERRAs, transcribed at chromosomes 1q-2q-4q-10q-13q-22q, 16p and 20q in healthy (n = 23) and pathological (n = 24) human endometrium and to examine their association with cellular proliferation, TA and telomere lengths. EC samples demonstrated significantly reduced levels of TERRAs for Chromosome 16p (Ch-16p) (p < 0.002) and Chromosome 20q (Ch-20q) (p = 0.0006), when compared with the postmenopausal samples. No significant correlation was found between TERRA levels and TA but both Ch-16p and Ch-20q TERRA levels negatively correlated with the proliferative marker Ki67 (r = -0.35, p = 0.03 and r = -0.42, p = 0.01 respectively). Evaluation of single telomere length analysis (STELA) at XpYp telomeres demonstrated a significant shortening in EC samples when compared with healthy tissues (p = 0.002). We detected TERRAs in healthy human endometrium and observed altered individual TERRA-specific levels in malignant endometrium. The negative correlation of TERRAs with cellular proliferation along with their significant reduction in EC may suggest a role for TERRAs in carcinogenesis and thus future research should explore TERRAs as potential therapeutic targets in EC.

Family and friends: Supporting oral care in care homes.

OBJECTIVE: To consider the role of family and friends in supporting oral care. BACKGROUND: People who live in care homes are susceptible to oral health problems, which can be detrimental to their health and personal and social well-being. External support from family members and friends has been indicated as being important for maintaining oral health for this vulnerable group of care home residents. MATERIALS AND METHODS: Qualitative one-to-one interviews were undertaken with care home residents, in Cardiff, UK. Further interviews were undertaken with care home personnel with responsibility for oral health care in order to contextualise residents' interview data. Interviews were audio recorded, transcribed and analysed using a thematic approach. RESULTS: A total of 26 interviews were conducted with care home residents and four interviews with care home personnel, across five care homes. Three main themes emanated from the data relating to co-supporting oral care: supplying oral care products; accessing dental care and enabling self-management of oral care problems. There were no spouse caregivers; family and friends acted as co-supporters of oral care providing a link to residents' pre-care home lives by informing the care home personnel of their relatives' normal routines. An overarching theme "balancing roles - maintaining the equilibrium" emerged from the data reflecting the roles that both care home personnel and family and friends had in balancing the needs, care and well-being of the resident. CONCLUSION: This study suggests that there are opportunities to improve oral health by providing support for family and friends of those people who are living in care, especially in relation to supplying oral care products, enabling self-management of oral care problems and accessing dental care.

Telomere length is a critical determinant for survival in multiple myeloma.

The variable clinical outcomes of Multiple Myeloma (MM) patients are incompletely defined by current prognostication tools. We examined the clinical utility of high-resolution telomere length analysis as a prognostic marker in MM. Cohort stratification, using a previously determined length threshold for telomere dysfunction, revealed that patients with short telomeres had a significantly shorter overall survival (P < 0·0001; HR = 3·4). Multivariate modelling using forward selection identified International Staging System (ISS) stage as the most important prognostic factor, followed by age and telomere length. Importantly, each ISS prognostic subset could be further risk-stratified according to telomere length, supporting the inclusion of this parameter as a refinement of the ISS.

Telomere length is an independent prognostic marker in MDS but not in de novo AML.

Telomere dysfunction is implicated in the generation of large-scale genomic rearrangements that drive progression to malignancy. In this study we used high-resolution single telomere length analysis (STELA) to examine the potential role of telomere dysfunction in 80 myelodysplastic syndrome (MDS) and 95 de novo acute myeloid leukaemia (AML) patients. Despite the MDS cohort being older, they had significantly longer telomeres than the AML cohort (P < 0·0001) where telomere length was also significantly shorter in younger AML patients (age <60 years) (P = 0·02) and in FLT3 internal tandem duplication-mutated AML patients (P = 0·03). Using a previously determined telomere length threshold for telomere dysfunction (3·81 kb) did not provide prognostic resolution in AML [Hazard ratio (HR) = 0·68, P = 0·2]. In contrast, the same length threshold was highly prognostic for overall survival in the MDS cohort (HR = 5·0, P < 0·0001). Furthermore, this telomere length threshold was an independent parameter in multivariate analysis when adjusted for age, gender, cytogenetic risk group, number of cytopenias and International Prognostic Scoring System (IPSS) score (HR = 2·27, P < 0·0001). Therefore, telomere length should be assessed in a larger prospective study to confirm its prognostic role in MDS with a view to integrating this variable into a revised IPSS.

Elaboration of tetra-orthogonally-substituted aromatic scaffolds towards novel EGFR-kinase inhibitors.

Nitration of three regioisomers of bromo-fluorobenzaldehyde proceeds regioselectively, notably with H2SO4/HNO3 at 0 °C. The thereby synthesized tetrasubstituted aromatics, endowed with orthogonal substituents, can be elaborated via Pd-catalysed coupling, reduction and reductive amination reactions. As a test-case, these compounds were converted into EGFR inhibitors related to Gefitinib, whose activity was rationalised by docking studies.

Telomere dysfunction accurately predicts clinical outcome in chronic lymphocytic leukaemia, even in patients with early stage disease.

Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI] = 11·6-106·4) and this was preserved in early-stage disease patients (P < 0·0001, HR=19·3, 95% CI = 17·8-802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL.

Mre11 modulates the fidelity of fusion between short telomeres in human cells.

The loss of telomere function can result in the fusion of telomeres with other telomeric loci, or non-telomeric double-stranded DNA breaks. Sequence analysis of fusion events between short dysfunctional telomeres in human cells has revealed that fusion is characterized by a distinct molecular signature consisting of extensive deletions and micro-homology at the fusion points. This signature is consistent with alternative error-prone end-joining processes. We have examined the role that Mre11 may play in the fusion of short telomeres in human cells; to do this, we have analysed telomere fusion events in cells derived from ataxia-telangiectasia-like disorder (ATLD) patients that exhibit hypomorphic mutations in MRE11. The telomere dynamics of ATLD fibroblasts were indistinguishable from wild-type fibroblasts and they were proficient in the fusion of short telomeres. However, we observed a high frequency of insertion of DNA sequences at the fusion points that created localized sequence duplications. These data indicate that Mre11 plays a role in the fusion of short dysfunctional telomeres in human cells and are consistent with the hypothesis that as part of the MRN complex it serves to stabilize the joining complex, thereby controlling the fidelity of the fusion reaction.

Maori Medical Student and Physician Exposure to Racism, Discrimination, Harassment, and Bullying.

Importance: Discrimination, bullying, and harassment in medicine have been reported internationally, but exposures for Indigenous medical students and physicians, and for racism specifically, remain less examined. Objective: To examine the prevalence of racism, discrimination, bullying, and harassment for Maori medical students and physicians in New Zealand and associations with demographic and clinical characteristics. Design, Setting, and Participants: This cross-sectional study used data from an anonymous national survey of Maori medical students and physicians in New Zealand in late 2021 and early 2022. Data were analyzed from March 2022 to April 2024. Exposures: Age, gender, marginalized status (ie, in addition to being Maori, belonging to other groups traditionally marginalized or underrepresented in medicine), year of medical school, year of graduation, and main work role. Main Outcomes and Measures: Direct and witnessed racism, discrimination, bullying, and harassment were measured as any experience in the last year and ever. Any exposure to negative comments about social groups and witnessing discriminatory treatment toward Maori patients or whanau (extended family). Considering leaving medicine, including because of mistreatment, was measured. Results: Overall, 205 Maori medical students (median [IQR] age, 23.1 [21.6-24.3] years; 137 [67.2%] women) and 200 physicians (median [IQR] age, 36.6 [30.1-45.3] years; 123 [62.8%] women) responded. Direct and witnessed exposure to racism (184 students [91.5%]; 176 physicians [90.7%]) and discrimination (176 students [85.9%]; 179 physicians [89.5%]) ever in medical education, training, or work environments was common. Ever exposure to witnessed and direct bullying (123 students [66.5%]; 150 physicians [89.3%]) and harassment (73 students [39.5%]; 112 physicians [66.7%]) was also common. Most respondents reported witnessing Maori patients or their whanau being treated badly in clinical settings, in direct interactions (67 students [57.8%]; 112 physicians [58.9%]) or behind their backs (87 students [75.0%]; 138 physicians [72.6%]). One-quarter of Maori medical students (45 students), and 37.0% of physicians (61 physicians) had considered leaving or taken a break from medicine because of these experiences. Additional marginalized statuses were significantly associated with any direct experience of mistreatment in the last year for students and physicians. Exposure to some forms of mistreatment were also significantly associated with higher likelihood of thinking about leaving or taking a break from medicine for physicians. Conclusions and Relevance: In this study, Maori medical students and physicians reported high exposure to multiple forms of racism, discrimination, bullying, and harassment in medical education, training, and work environments, requiring an urgent response from medical institutions.