Live-cell fluorescence imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum.

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development.

Care challenges of home health patients living with dementia: a pathway forward with palliative care.

BACKGROUND: Home health care (HHC) is a leading source of care support for older adults with serious illness, particularly patients living with dementia (PLWD). Demand for HHC is expected to continue to grow, driven by an aging population and preference for non-institutional care. HHC agencies are frequently under pressure to find effective approaches for improving care delivery and quality. One strategy that has the potential to improve the quality of life and patient satisfaction in HHC for PLWD is the integration of palliative care. Therefore, we sought to understand the experiences and needs of PLWD and their family caregivers specifically focusing on ways that HHC and palliative care may be integrated as part of the care transition from hospital to home, to better support PLWD and their families. METHODS: We conducted a descriptive qualitative study focusing on the perspectives of patients, caregivers, and healthcare team members about palliative care delivery for patients receiving HHC. Interviews were audio-recorded and professionally transcribed. In this analysis, we specifically report on dementia-related content using an iterative, team-based thematic analysis approach. RESULTS: We identified three themes: 1) 'Living in the Whirlwind' which describes the many competing demands on caregivers time and the associated feeling of loss of control, 2) 'Thinking Ahead' which describes the importance of thinking beyond the day-to-day tasks to begin planning for the future, and 3) 'Pathways Forward' which describes the integration of palliative care into HHC to provide enhanced support for PLWD and their caregivers. CONCLUSION: In this qualitative study, our formative work identified the importance of providing anticipatory guidance (e.g., safety, advance care planning) coupled with emotional and pragmatic care supports (e.g., finding resources, navigating insurance) to sustain caregivers who are struggling with the whirlwind.

"Our Hands Are Tied Until Your Doctor Gets Here": Nursing Perspectives on Inter-hospital Transfers.

BACKGROUND: The transfer of patients between hospitals (inter-hospital transfer, or IHT) is a common occurrence for patients, but guidelines to ensure safe and effective IHTs are lacking. Poor IHTs result in higher rates of mortality, longer lengths of stay, and higher hospitalization costs compared to admissions from the emergency department. Nurses are often the first point of contact for IHT patients and can provide valuable insights on key challenges to IHT processes. OBJECTIVE: To characterize the experiences of inpatient floor-level bedside nurses caring for IHT patients and identify care coordination challenges and solutions. DESIGN/PARTICIPANTS/APPROACH: Qualitative study using semi-structured focus groups and interviews conducted from October 2019 to July 2020 with 21 inpatient floor-level nurses caring for adult medicine patients at an academic hospital. Nurses were recruited using a purposive convenience sampling approach. A combined inductive and deductive coding approach guided by thematic analysis was used for data analysis. KEY RESULTS: Results from this study are mapped to the Agency for Healthcare Research and Quality Care Coordination Measurement Framework domains of communication, assessing needs and goals, and negotiating accountability. The following key themes characterize nurses' experiences with IHT related to these domains: (1) challenges with information exchange and team communication during IHT, (2) environmental and information preparation needed to anticipate transfers, and (3) determining responsibility and care plans after the IHT patient has arrived at the accepting facility. CONCLUSIONS: Nurses described the absence of standardized processes to coordinate care before or at the time of patient arrival. Challenges to communication and coordination during IHTs negatively impacted patient care and nursing professional satisfaction. To streamline care for IHT patients and reduce nursing stress, future IHT interventions should include standardized handoff reports, timely identification and easy access to admitting clinicians, and timely clinician evaluation and orders.

4E-BP1 Protects Neurons from Misfolded Protein Stress and Parkinson's Disease Toxicity by Inducing the Mitochondrial Unfolded Protein Response.

Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Overexpression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition on mTORC1 repression may be neuroprotective; however, whether 4E-BP1 overexpression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1-overexpressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1-overexpressing primary neurons. After documenting that 4E-BP1-overexpressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins (rotenone, maneb, or paraquat) and documented significant protection in neurons from newborn male and female 4E-BP1-OE transgenic mice. We observed 4E-BP1-dependent upregulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 overexpression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested whether 4E-BP1 could prevent α-synuclein neurotoxicity by treating 4E-BP1-overexpressing primary neurons with α-synuclein preformed fibrils, and we observed marked reductions in α-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD.SIGNIFICANCE STATEMENT In neurodegenerative disease, misfolded proteins accumulate and overwhelm normal systems of homeostasis and quality control. One mechanism for improving protein quality control is to reduce protein translation. Here we investigated whether neuronal overexpression of 4E-BP1, a key repressor of protein translation, can protect against misfolded protein stress and toxicities linked to Parkinson's disease, and found that 4E-BP1 overexpression prevented cell death in neurons treated with brefeldin A, rotenone, maneb, paraquat, or preformed fibrils of α-synuclein. When we sought the basis for 4E-BP1 neuroprotection, we discovered that 4E-BP1 activation promoted the mitochondrial unfolded protein response. Our findings highlight 4E-BP1 as a therapeutic target in neurodegenerative disease and underscore the importance of the mitochondrial unfolded protein response in neuroprotection against various insults.

Resident perspectives on the value of interdisciplinary conference calls for geriatric patients.

BACKGROUND: There are limited competency-based educational curricula for transitions of care education (TOC) for internal medicine (IM) residency programs. The University of Colorado implemented a virtual interdisciplinary conference call, TEAM (Transitions Expectation and Management), between providers on the inpatient Acute Care of the Elder (ACE) unit and the outpatient Seniors Clinic at the University of Colorado Hospital. Residents rotating on the ACE unit participated in weekly conferences discussing Seniors Clinic patients recently discharged, or currently hospitalized, to address clinical concerns pertaining to TOC. Our goals were to understand resident perceptions of the educational value of these conferences, and to determine if these experiences changed attitudes or practice related to care transitions. METHODS: We performed an Institutional Review Board-approved qualitative study of IM housestaff who rotated on the ACE unit during 2018-2019. Semi-structured interviews were conducted to understand perceptions of the value of TEAM calls for residents' own practice and the impact on patient care. Data was analyzed inductively, guided by thematic analysis. RESULTS: Of the 32 IM residents and interns who rotated on ACE and were invited to participate, 11 agreed to an interview. Three key themes emerged from interviews that highlighted residents' experiences identifying and navigating some of their educational 'blind spots:' 1) Awareness of patient social complexities, 2) Bridging gaps in communication across healthcare settings, 3) Recognizing the value of other disciplines during transitions. CONCLUSIONS: This study highlights learner perspectives of the benefit of interdisciplinary conference calls between inpatient and outpatient providers to enhance transitions of care, which provide meaningful feedback and serve as a vehicle for residents to recognize the impact of their care decisions in the broader spectrum of patients' experience during hospital discharge. Educators can maximize the value of these experiences by promoting reflective debriefs with residents and bringing to light previously unrecognized knowledge gaps around hospital discharge.

Perceptions of trained laypersons in end-of-life or advance care planning conversations: a qualitative meta-synthesis.

BACKGROUND: Laypersons including volunteers, community health navigators, or peer educators provide important support to individuals with serious illnesses in community or healthcare settings. The experiences of laypersons in communication with seriously ill peers is unknown. METHODS: We performed an ENTREQ-guided qualitative meta-synthesis. We conducted a systematic search of MEDLINE, PsycINFO, CINAHL, Cochrane Library, and AMED to include qualitative studies with data regarding communication and laypersons in advance care planning, palliative care, or end-of-life settings. Study quality was appraised using a standardized tool. The analysis identified key domains and associated themes relating specifically to laypersons' perspectives on communication. RESULTS: Of 877 articles, nine studies provided layperson quotations related to layperson-to-peer communication associated with advance care planning (n = 4) or end-of-life conversations (n = 5). The studies were conducted in United Kingdom (n = 4) or United States settings (n = 5). The synthesis of layperson perspectives yielded five main domains: 1) layperson-to-peer communication, focusing on the experience of talking with peers, 2) layperson-to-peer interpersonal interactions, focusing on the entire interaction between the layperson and peers, excluding communication-related issues, 3) personal impact on the layperson, 4) layperson contributions, and 5) layperson training. Laypersons described using specific communication skills including the ability to build rapport, discuss sensitive issues, listen and allow silence, and respond to emotions. CONCLUSIONS: Published studies described experiences of trained laypersons in conversations with peers related to advance care planning or end-of-life situations. Based on these layperson perspectives related to communication, programs should next evaluate the potential impact of laypersons in meaningful conversations.

"We're all truly pulling in the exact same direction": A qualitative study of attending and resident physician impressions of structured bedside interdisciplinary rounds.

BACKGROUND: Physicians often hold leadership roles in implementing interdisciplinary rounds (IDR) in clinical settings, thus understanding physician perspectives of bedside IDR is crucial. OBJECTIVE: To understand physicians' perspectives of structured bedside IDR. DESIGN: Qualitative descriptive study. SETTING AND PARTICIPANTS: Medical and transitional year resident and hospitalist physicians participating in structured bedside IDR in a community teaching hospital affiliated with a large academic center. METHODS: We conducted semi-structured interviews and focus groups with study participants about their experiences with participating in structured bedside IDR between July 2017 and April 2018. All interviews were recorded and professionally transcribed. We used thematic analysis to identify key themes. RESULTS: We interviewed 41 resident physicians and 10 hospitalist physicians and discovered five key themes that highlight physician impressions of bedside IDR. Bedside IDR decreased unnecessary care, screened for risks/errors, created a shared mental model of care, and increased physician empathy for the patient and the interprofessional care team. Physicians felt patients valued the streamlined communication they experienced but questioned the intrusiveness of large healthcare teams. Challenges to bedside IDR included the meaningful engagement of key stakeholders and, particularly for resident physicians, the difficultly of managing unstandardized communication in a standardized time-sensitive setting. CONCLUSIONS: Structured bedside IDR have the capacity to improve patient care, interprofessional teamwork, and physician empathy for patients and the interprofessional team. However, sustained leadership support and clearly defined rounds goals and objectives are necessary ingredients to maximize the benefits and address the challenges of bedside IDR.

A Mixed-Methods Comparison of Interventions to Increase Advance Care Planning.

PURPOSE: Although interventions can increase advance care planning (ACP) engagement, it remains unclear which interventions to choose in primary care settings. This study compares a passive intervention (mailed materials) to an interactive intervention (group visits) on participant ACP engagement and experiences. METHODS: We used mixed methods to examine ACP engagement at baseline and six months following two ACP interventions. Eligible patients were randomized to receive mailed materials or participate in two ACP group visits. We administered the 4-item ACP Engagement survey (n = 110) and conducted interviews (n = 23). We compared mean scores and percent change in ACP engagement, analyzed interviews with directed content analysis to understand participants' ACP experiences, and integrated the findings based on mailed materials or group visits intervention. RESULTS: All participants demonstrated increased ACP engagement scores. At six months, group visit participants reported higher percent change in mean overall score compared with mailed materials participants (+8% vs +3%, P < .0001). Group visits participants reported that being prompted to think about end-of-life preferences, gaining knowledge about ACP, and understanding the value of completing ACP documentation influenced their ACP readiness. While both interventions encouraged patients to start considering and refining their end-of-life preferences, group visits made patients feel more knowledgeable about ACP, highlighted the importance of completing ACP documentation early, and sparked further ACP discussions with others. CONCLUSIONS: While primary care patients may benefit from mailed ACP materials, patients reported increased readiness after ACP group visits. Group visits emphasized the value of upstream preparation, ongoing conversations, and increased knowledge about ACP.

Holding back in communications with clinicians: Patient and care partner perspectives in Parkinson's disease.

Objectives: To describe how patients with Parkinson's disease (PD) and care partners choose to share or withhold information from clinicians. Methods: This is a qualitative, descriptive study nested within a multisite, randomized clinical trial of outpatient palliative care compared to standard neurologic care for PD. Interviews with patients (n = 30) and care partners (n = 30) explored experiences communicating with neurology clinicians. Thematic analyses identified themes relevant to patient-care partner-clinician communication. Results: There were four themes relevant to sharing and/or withholding information from clinicians: (1) Suppressing Concerns During Visits, (2) Care Partner Awareness of Patients' Communication Barriers due to Cognitive Impairment, (3) Limited Sharing of Sensitive or Intimate Issues by Patients and Care Partners, and (4) Patient and Care Partner Suggestions to Overcome 'Holding Back'. Conclusion: Limitations to communication between patients, care partners, and clinicians should be acknowledged and recognized in routine Parkinson's disease care to foster accurate disclosure of unmet palliative care and other needs. Triadic communication strategies may help patients and care partners talk about unmet palliative care needs. Innovation: By recognizing that cognitive impairment and sensitive topics can be barriers, clinicians can adjust or adopt targeted communication strategies for identifying and discussing care needs.

Palliative Care across Settings: Perspectives from Inpatient, Primary Care, and Home Health Care Providers and Staff.

BACKGROUND: Early introduction of palliative care can improve patient-centered outcomes for older adults with complex medical conditions. However, identifying the need for and introducing palliative care with patients and caregivers is often difficult. We aim to identify how and why a multi-setting approach to palliative care discussions may improve the identification of palliative care needs and how to facilitate these conversations. METHODS: Descriptive qualitative study to inform the development and future pilot testing of a model to improve recognition of, and support for, unmet palliative care needs in home health care (HHC). Thematic analysis of semi-structured interviews with providers across inpatient (n = 11), primary care (n = 17), and HHC settings (n = 10). RESULTS: Four key themes emerged: 1) providers across settings can identify palliative care needs using their unique perspectives of the patient's care, 2) identifying palliative care needs is challenging due to infrequent communication and lack of shared information between providers, 3) importance of identifying a clinical lead of patient care who will direct palliative care discussions (primary care provider), and 4) importance of identifying a care coordination lead (HHC) to bridge communication among multi-setting providers. These themes highlight a multi-setting approach that would improve the frequency and quality of palliative care discussions. CONCLUSIONS: A lack of structured communication across settings is a major barrier to introducing and providing palliative care. A novel model that improves communication and coordination of palliative care across HHC, inpatient and primary care providers may facilitate identifying and addressing palliative care needs in medically complex older adults.

A novel class of sulphonamides potently block malaria transmission by targeting a Plasmodium vacuole membrane protein.

Phenotypic cell-based screens are critical tools for discovering candidate drugs for development, yet identification of the cellular target and mode of action of a candidate drug is often lacking. Using an imaging-based screen, we recently discovered an N-[(4-hydroxychroman-4-yl)methyl]-sulphonamide (N-4HCS) compound, DDD01035881, that blocks male gamete formation in the malaria parasite life cycle and subsequent transmission of the parasite to the mosquito with nanomolar activity. To identify the target(s) of DDD01035881, and of the N-4HCS class of compounds more broadly, we synthesised a photoactivatable derivative, probe 2. Photoaffinity labelling of probe 2 coupled with mass spectrometry identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as a potential parasite target. Complementary methods including cellular thermal shift assays confirmed that the parent molecule DDD01035881 stabilised Pfs16 in lysates from activated mature gametocytes. Combined with high-resolution, fluorescence and electron microscopy data, which demonstrated that parasites inhibited with N-4HCS compounds phenocopy the targeted deletion of Pfs16 in gametocytes, these data implicate Pfs16 as a likely target of DDD01035881. This finding establishes N-4HCS compounds as being flexible and effective starting candidates from which transmission-blocking antimalarials can be developed in the future.

Frontline healthcare workers' perspectives on interprofessional teamwork during COVID-19.

Effective interprofessional teamwork serves an important role in successful crisis response. Responses to past public health crises have relied on interprofessional communication and trust to support healthcare worker (HCW) resiliency. To understand interprofessional interactions and perceptions of teamwork during the COVID-19 response, we conducted in-depth semi-structured interviews with 18 inpatient HCWs (11 bedside nurses, 5 care coordinators, and 2 pharmacists) from one VA Medical Center between March and June 2020. Using thematic analysis, we identified four key themes that describe the strengths and challenges of interprofessional teamwork, communication, patient care, and organizational response during the initial COVID-19 surge. Interprofessional teams were fragmented. HCWs who transitioned to remote work lost their status on inpatient teams and struggled to provide pre-pandemic levels of quality of care. Conversely, interprofessional teamwork improved for HCWs who continued to work on inpatient units, where study participants described a decline in interprofessional hierarchies and an increase in mutual support. Participants described the need for timely, accurate, transparent communication as they faced new patient safety and communication challenges brought on by the pandemic. HCWs expressed a desire for sustained leadership support and inclusion in institutional decision-making. The challenges to teamwork, communication, and patient care reported in this study highlight the need for consistent, transparent communication and organizational response from hospital leadership during times of crisis.

Forgotten frontline workers: Environmental health service employees' perspectives on working during the COVID-19 pandemic.

BACKGROUND: Environmental Health Service employees (EVS) sanitize healthcare facilities and are critical to preventing infection, but are under-resourced during the COVID-19 pandemic and at risk of burnout. OBJECTIVE: Understand demands on EVS' work and strain on resources during COVID-19. DESIGN: Qualitative descriptive study conducted in winter 2020-2021. SETTING: One quaternary care academic medical center in Colorado. PARTICIPANTS: A convenience sample of 16 EVS out of 305 eligible at the medical center. Fifty percent identified as Black, 31% as Hispanic, 6% as Asian, and 6% as White (another 6% identified as mixed race). Sixty-nine percent were female, and half were born in a country outside the United States. MEASURES: Semistructured telephone interviews. Interviews were audio-recorded and transcribed, and thematic analysis was used to identify key themes. RESULTS: Four themes illustrate EVS experiences with job strain and support during COVID-19: (1) Needs for ongoing training/education, (2) Emotional challenges of patient care, (3) Resource/staffing barriers, and (4) Lack of recognition as frontline responders. Despite feeling unrecognized during the pandemic, EVS identified structural supports with potential to mitigate job strain, including opportunities for increased communication with interdisciplinary colleagues, intentional acknowledgment, and education for those who speak languages other than English. Strategies that can increase physical and emotional resources and reduce job demands have potential to combat EVS burnout. CONCLUSIONS: As the surge of COVID-19 cases continues to overwhelm healthcare facilities, healthcare systems and interdisciplinary colleagues can adopt policies and practices that ensure lower-wage healthcare workers have access to resources, education, and emotional support.

Care planning priorities of older patients with advanced bladder cancer.

OBJECTIVES: Advanced bladder cancer (ABC) disproportionately affects older adults, and little is known about older patients' priorities for care planning in advanced cancer. Patient-centered communication remains crucial to shared decision-making between patients, families, and providers. Yet, older patients with cancer may not always know how to express their preferences, and oncologists do not always review patients' informational needs. This study aimed to understand preferences of older patients with ABC related to their communication with providers and navigation of care planning. MATERIALS AND METHODS: This qualitative descriptive study involved in-depth interviews and focus groups with older patients with ABC and their care partners, which explored their priorities for care planning and communication with providers, decision-making processes, and valued traits in ABC care. Data were analyzed using thematic analysis. RESULTS: Ten participants attended focus groups or interviews. Seven patients were male and three care partners were female. The mean age was 74. Time since ABC diagnosis ranged from three to seventeen months. Four key themes illustrate participants' priorities in their ABC care as older adults: 1. The significance of key phrasing in communication from oncologists, 2. The need for clear expectation-setting about prognosis and treatment, 3. The role of others in patient care decisions, and 4. Valued traits in care communication. CONCLUSION: Older patients with ABC and their care partners are active participants in their care. Oncologists should prioritize setting clear expectations for treatment, involving family in care planning, and communicating honestly about expected changes to quality of life and functional status.

Relationship between systems-level factors and hand hygiene adherence.

This study was a cross-sectional descriptive survey of acute care hospitals in California to describe staff hand hygiene compliance and related predictors and explore the relationship between hand hygiene adherence and health care-associated infections. Although there was a relatively small sample size, institutions with morning huddles reported a significantly higher proportion of 95% or more hand hygiene compliance. Huddles are an organizational tool to improve teamwork and communication and may offer promise to influence hand hygiene adherence.

Methotrexate pneumonitis in rheumatoid arthritis: increased prevalence with increasing latitude: an epidemiological study of trends in new zealand.

BACKGROUND: There is an association between increasing prevalence and increasing latitude for some autoimmune diseases, including rheumatoid arthritis (RA). Furthermore, in RA patients, a geographical variation in methotrexate pneumonitis has been suggested at a regional level in New Zealand. OBJECTIVE: The objective of the study was to determine if there is an increased incidence of methotrexate pneumonitis with increasing latitude in New Zealand. METHODS: A search was conducted using the NZ Ministry of Health's National Minimum Data Set for patients with discharge codes for RA (M05, M06) or history of RA and drug-induced lung disease (J702, J703, J704) or other (J189, J680, J90, J984) and methotrexate (Y431), for the period July 1, 1999, to June 30, 2008. Anonymous data were provided by the Ministry of Health for the 43 patients fulfilling these coding criteria and also the latitude and population of each domicile code. A Poisson regression analysis was undertaken with latitude as a continuous variable, adjusting for the total population at different latitudes. RESULTS: The incidence rate ratio for methotrexate pneumonitis shows a 16% increase per 1 degree of latitude (95% confidence interval, 7%-27%; P = 0.02). CONCLUSIONS: There was a latitudinal gradient seen in the rate of patient discharges for methotrexate pneumonitis, in the defined period. This supports the hypothesis that there is a latitude-dependent risk factor for this disorder and raises questions regarding possible environmental cofactors. It also supports the growing pool of evidence that certain immune-mediated conditions are more common at higher latitudes.

Predictive markers of anthracycline benefit: a prospectively planned analysis of the UK National Epirubicin Adjuvant Trial (NEAT/BR9601).

BACKGROUND: The NEAT/BR9601 trial showed benefit for addition of anthracyclines to cyclophosphamide, methotrexate, and fluorouracil (CMF) as adjuvant treatment for early breast cancer. We investigated prospectively predictive biomarkers of anthracycline benefit including HER2 and TOP2A. METHODS: 1941 tumours from 2391 women recruited to NEAT/BR9601 were analysed on tissue microarrays for HER2 and TOP2A amplification and deletion, HER1-3 and Ki67 expression, and duplication of chromosome 17 centromere enumeration probe (Ch17CEP). Log-rank analyses identified factors affecting relapse-free and overall survival, and regression models tested independent prognostic effect of markers, with adjustment for known prognostic factors (age, nodal status, oestrogen-receptor status, grade, and tumour size). The predictive value of markers was tested by treatment interactions for relapse-free and overall survival. FINDINGS: 1762 patients were analysed. 21% of tumours (n=367) were HER2 amplified, 10% were TOP2A amplified (n=169), 11% showed TOP2A deleted (n=191), 23% showed Ch17CEP duplication (n=406), and 61% had high (>13.0%) Ki67 (n=1136). In univariate analyses, only HER2 amplification and TOP2A deletion were significant prognostic factors for relapse-free (hazard ratio [HR] 1.59, 95% CI 1.32-1.92, p<0.0001; and 1.52, 1.20-1.92, p=0.0006, respectively) and overall survival (1.79, 1.47-2.19, p<0.0001; and 1.62, 1.26-2.08, p=0.0002 respectively). We detected no significant interaction with anthracycline benefit for Ki67, HER2, HER1-3, or TOP2A. By contrast, in multivariate analyses, Ch17CEP duplication was associated with significant improvements in both relapse-free (HR 0.92, 95% CI 0.72-1.18 for tumours with normal Ch17CEP vs 0.52, 0.34-0.81 for tumours with abnormal Ch17CEP; p for interaction=0.004) and overall survival (0.94, 0.72-1.24 vs 0.57, 0.36-0.92; p for interaction=0.02) with anthracycline use. INTERPRETATION: In women with early breast cancer receiving adjuvant chemotherapy, the most powerful predictor of benefit from anthracyclines is Ch17CEP duplication. In view of the location of HER2/TOP2A on chromosome 17, Ch17CEP duplication might explain the inconsistencies in previous studies of factors predicting benefit from anthracyclines. FUNDING: Cancer Research UK and the Scottish Breast Cancer Clinical Trials Group.

Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum.

Recent studies highlight the emerging role of lipids as important messengers in malaria parasite biology. In an attempt to identify interacting proteins and regulators of these dynamic and versatile molecules, we hypothesised the involvement of phospholipid translocases and their substrates in the infection of the host erythrocyte by the malaria parasite Plasmodium spp. Here, using a data base searching approach of the Plasmodium Genomics Resources (www.plasmodb.org), we have identified a putative phospholipid (PL) scramblase in P. falciparum (PfPLSCR) that is conserved across the genus and in closely related unicellular algae. By reconstituting recombinant PfPLSCR into liposomes, we demonstrate metal ion dependent PL translocase activity and substrate preference, confirming PfPLSCR as a bona fide scramblase. We show that PfPLSCR is expressed during asexual and sexual parasite development, localising to different membranous compartments of the parasite throughout the intra-erythrocytic life cycle. Two different gene knockout approaches, however, suggest that PfPLSCR is not essential for erythrocyte invasion and asexual parasite development, pointing towards a possible role in other stages of the parasite life cycle.

Post-Covid-19 Syndrome: Improvements in Health-Related Quality of Life Following Psychology-Led Interdisciplinary Virtual Rehabilitation.

Coronavirus disease 2019 (COVID-19) is increasingly recognized as having significant long-term impact on physical and mental health. The Primary Care Wellbeing Service (PCWBS) in Bradford District Care NHS Foundation Trust (BDCFT) is a psychology-led specialist interdisciplinary team of health professionals specializing in persistent physical symptoms (PPS) and Chronic Fatigue Syndrome (CFS)/Myalgic Encephalomyelitis (ME) with an emphasis on holistic integrated care. The PCWBS quickly recognized the risk of the long-term effects of COVID-19, particularly for social, health and care staff, and developed a 7-week virtual rehabilitation course which was piloted in October 2020. The "Recovering from COVID" course takes a whole system, biopsychosocial approach to understanding COVID-19 and post-viral fatigue (PVF) and is delivered by an interdisciplinary team consisting of a clinical psychologist, physiotherapist, occupational therapist, dietitian, speech and language therapist, assistant psychologist, and a personal support navigator with support from a team administrator. The course focuses on understanding PVF, sleep optimization, nutrition, swallowing, activity management, energy conservation, stress management, breathing optimization, managing setbacks, and signposting to appropriate resources and services. Since the pilot, PCWBS has delivered 7 courses to support over 200 people suffering from post-COVID-19 syndrome. One hundred and forty-nine individuals that enrolled on the "Recovering from COVID" course completed the EQ-5D-5L to assess Health-related quality of life (HRQoL) across 5 dimensions, including problems with mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Subsequently, 76 individuals completed these measures at the end of the rehabilitation course showing that patient ratings were significantly improved. In response to the NIHR recommendation for rapid evaluation of different service models for supporting people with post-COVID-19 syndrome, this data offers hope that rehabilitation is effective in reversing some of the problems faced by people living with the long-term effects of COVID-19.

The relative toxicity of compounds used as preservatives in vaccines and biologics.

BACKGROUND: In vaccines/biologics, preservatives are used to prevent microbial growth. MATERIAL/METHODS: The present study examined: (1) the comparative toxicities of commonly used preservatives in US licensed vaccines to human neurons; and (2) the relative toxicity index of these compounds to human neurons in comparison to bacterial cells. RESULTS: Using human neuroblastoma cells, the relative cytotoxicity of the levels of the compounds commonly used as preservative in US licensed vaccines was found to be phenol <2-phenoxyethanol < benzethonium chloride < Thimerosal. The observed relative toxicity indices (human neuroblastoma cells/bacterial cells) were 2-phenoxyethanol (4.6-fold) < phenol (12.2-fold) < Thimerosal (>330-fold). In addition, for the compounds tested, except for 2-phenoxyethanol, the concentrations necessary to induce significant killing of bacterial cells were significantly higher than those routinely present in US licensed vaccine/biological preparations. CONCLUSIONS: None of the compounds commonly used as preservatives in US licensed vaccine/biological preparations can be considered an ideal preservative, and their ability to fully comply with the requirements of the US Code of Federal Regulations (CFR) for preservatives is in doubt. Future formulations of US licensed vaccines/biologics should be produced in aseptic manufacturing plants as single dose preparations, eliminating the need for preservatives and an unnecessary risk to patients.