DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

Psychosocial outcomes and health service use after notifying women participating in population breast screening when they have dense breasts: a BreastScreen Queensland randomised controlled trial.

BACKGROUND: Robust evidence regarding the benefits and harms of notifying Australian women when routine breast screening identifies that they have dense breasts is needed for informing future mammography population screening practice and policy. OBJECTIVES: To assess the psychosocial and health services use effects of notifying women participating in population-based breast cancer screening that they have dense breasts; to examine whether the mode of communicating this information about its implications (print, online formats) influences these effects. METHODS AND ANALYSIS: The study population comprises women aged 40 years or older who attend BreastScreen Queensland Sunshine Coast services for mammographic screening and are found to have dense breasts (BI-RADS density C or D). The randomised controlled trial includes three arms (952 women each): standard BreastScreen care (no notification of breast density; control arm); notification of dense breasts in screening results letter and print health literacy-sensitive information (intervention arm 1) or a link or QR code to online video-based health literacy-sensitive information (intervention arm 2). Baseline demographic data will be obtained from BreastScreen Queensland. Outcomes data will be collected in questionnaires at baseline and eight weeks, twelve months, and 27 months after breast screening. Primary outcomes will be psychological outcomes and health service use; secondary outcomes will be supplemental screening outcomes, cancer worry, perceived breast cancer risk, knowledge about breast density, future mammographic screening intentions, and acceptability of notification about dense breasts. ETHICS APPROVAL: Gold Coast Hospital and Health Service Ethics Committee (HREC/2023/QGC/89770); Sunshine Coast Hospital and Health Service Research Governance and Development (SSA/2023/QSC/89770). DISSEMINATION OF FINDINGS: Findings will be reported in peer-reviewed journals and at national and international conferences. They will also be reported to BreastScreen Queensland, BreastScreen Australia, Cancer Australia, and other bodies involved in cancer care and screening, including patient and support organisations. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12623000001695p (prospective: 9 January 2023).

Force-Dependent Interactions between Talin and Full-Length Vinculin.

Talin and vinculin are part of a multicomponent system involved in mechanosensing in cell-matrix adhesions. Both exist in autoinhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talin's interaction with vinculin has not been investigated. Here by quantifying the kinetics of force-dependent talin-vinculin interactions using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin, resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an autoinhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

Negotiated Sharing of Pandemic Data, Models, and Resources.

Urgent responses to the COVID-19 pandemic depend on increased collaboration and sharing of data, models, and resources among scientists and researchers. In many scientific fields and disciplines, institutional norms treat data, models, and resources as proprietary, emphasizing competition among scientists and researchers locally and internationally. Concurrently, long-standing norms of open data and collaboration exist in some scientific fields and have accelerated within the last two decades. In both cases-where the institutional arrangements are ready to accelerate for the needed collaboration in a pandemic and where they run counter to what is needed-the rules of the game are "on the table" for institutional-level renegotiation. These challenges to the negotiated order in science are important, difficult to study, and highly consequential. The COVID-19 pandemic offers something of a natural experiment to study these dynamics. Preliminary findings highlight: the chilling effect of politics where open sharing could be expected to accelerate; the surprisingly conservative nature of contests and prizes; open questions around whether collaboration will persist following an inflection point in the pandemic; and the strong potential for launching and sustaining pre-competitive initiatives.

TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis.

The timing of cell division is controlled by the coupled regulation of growth and division. The target of rapamycin (TOR) signalling network synchronises these processes with the environmental setting. Here, we describe a novel interaction of the fission yeast TOR complex 2 (TORC2) with the cytokinetic actomyosin ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that the myosin II protein Myp2 and the myosin V protein Myo51 play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2-dependent phosphorylation of actin-capping protein 1 (Acp1, a known regulator of cytokinesis) controls CAR stability, modulates Acp1-Acp2 (the equivalent of the mammalian CAPZA-CAPZB) heterodimer formation and is essential for survival upon stress. Thus, TORC2 localisation to the CAR, and TORC2-dependent Acp1 phosphorylation contributes to timely control and the fidelity of cytokinesis and cell division.

Review and Comparison of Electronic Patient-Facing Family Health History Tools.

Family health history (FHx) is one of the most important pieces of information available to help genetic counselors and other clinicians identify risk and prevent disease. Unfortunately, the collection of FHx from patients is often too time consuming to be done during a clinical visit. Fortunately, there are many electronic FHx tools designed to help patients gather and organize their own FHx information prior to a clinic visit. We conducted a review and analysis of electronic FHx tools to better understand what tools are available, to compare and contrast to each other, to highlight features of various tools, and to provide a foundation for future evaluation and comparisons across FHx tools. Through our analysis, we included and abstracted 17 patient-facing electronic FHx tools and explored these tools around four axes: organization information, family history collection and display, clinical data collected, and clinical workflow integration. We found a large number of differences among FHx tools, with no two the same. This paper provides a useful review for health care providers, researchers, and patient advocates interested in understanding the differences among the available patient-facing electronic FHx tools.

Implementing Group Prenatal Care in Southwest Georgia Through Public-Private Partnerships.

Introduction CenteringPregnancy® is well-regarded as an innovative group model of prenatal care. In 2009, Georgia's Southwest Public Health District partnered with local obstetricians and medical centers to expand prenatal care access and improve perinatal outcomes for low-income women by implementing Georgia's first public health administered CenteringPregnancy program. This paper describes the successful implementation of CenteringPregnancy in a public health setting with no prior prenatal services; assesses the program's first 5-year perinatal outcomes; and discusses several key lessons learned. Methods Prenatal and hospital medical records of patients were reviewed for the time period from October 2009 through October 2014. Descriptive analyses were conducted to examine demographic and clinical characteristics of women initiating prenatal care and to assess perinatal outcomes among patients with singleton live births who attended at least three CenteringPregnancy sessions or delivered prior to attending the third session. Results Six hundred and six low-income women initiated prenatal care; 55.4 and 36.4% self-identified as non-Hispanic black and Hispanic, respectively. The median age was 23 years (IQR 20, 28). Nearly 69% initiated prenatal care in the first trimester. Perinatal outcomes were examined among 338 singleton live births. The 2010-2014 preterm birth rate (% of births < 37 weeks gestation at delivery) and low birth weight rate (% of births < 2500 g) were 9.1 and 8.9%, respectively. Nearly 77% of women initiated breastfeeding. Discussion CenteringPregnancy administered via public-private partnership may improve access to prenatal care and perinatal outcomes for medically underserved women in low-resource settings.

Measuring arm function early after stroke: is the DASH good enough?

OBJECTIVE: Despite a growing call to use patient-reported outcomes in clinical research, few are available for measuring upper limb function post-stroke. We examined the Disabilities of the Arm, Shoulder and Hand (DASH) to evaluate its measurement performance in acute stroke. In doing so, we compared results from traditional and modern psychometric methods. METHODS: 172 people with acute stroke completed the DASH. Those with upper limb impairments completed the DASH again at 6 weeks (n=99). Data (n=271) were analysed using two psychometric paradigms: traditional psychometric (Classical Test Theory, CTT) analyses examined data completeness, scaling assumptions, targeting, reliability and responsiveness; Rasch Measurement Theory (RMT) analyses examined scale-to-sample targeting, scale performance and person measurement. RESULTS: CTT analyses implied the DASH was psychometrically robust in this sample. Data completeness was high, criteria for scaling assumptions were satisfied (item-total correlations 0.55-0.95), targeting was good, internal consistency reliability was high (Cronbach's α=0.99) and responsiveness was clinically moderate (effect size=0.51). However, RMT analyses identified important limitations: scale-to-sample targeting was suboptimal, 4 items had disordered response category thresholds, 16 items exhibited misfit, 3 pairs of items had high residual correlations (>0.60) and 84 person fit residuals exceeded the recommended range. CONCLUSIONS: RMT methods identified limitations missed by CTT and indicate areas for improvement of the DASH as an upper limb measure for acute stroke. Findings, similar to those identified in multiple sclerosis, highlight the need for scales to have strong conceptual underpinnings, with their development and modification guided by sophisticated psychometric methods.

Regional Variation in Diabetic Outcomes by Country-of-Origin and Language in an Urban Safety Net Hospital.

Adherence to diabetic care guidelines among US immigrants remains low. This study assesses adherence to diabetic care guidelines by country-of-origin and language among a limited English-proficient (LEP) population. Timely completion of diabetic measures and acceptable levels of hemoglobin A1c (A1c), low density lipoprotein (LDL) cholesterol, and blood pressure (BP) were compared between LEP and English-proficient (EP) patients in this 2013 retrospective cohort study of adult diabetics. More LEP patients met BP targets (83 vs. 68 %, p < 0.0001) and obtained LDL targets (89 vs. 85 %, p = 0.0007); however, they had worse LDL control (57 vs. 62 %, p = 0.0011). Ethiopians and Somalians [adjusted OR (95 % CI) = 0.44 (0.30, 0.63)] were less likely than Latin Americans to meet BP goals. LEP patients outperformed EP peers on several diabetic outcomes measures with important variation between groups. These data highlight the success of a safety net hospital in improving diabetes management among diverse populations.

Discovery of a novel Kv7 channel opener as a treatment for epilepsy.

Facilitating activation, or delaying inactivation, of the native Kv7 channel reduces neuronal excitability, which may be beneficial in controlling spontaneous electrical activity during epileptic seizures. In an effort to identify a compound with such properties, the structure-activity relationship (SAR) and in vitro ADME for a series of heterocyclic Kv7.2-7.5 channel openers was explored. PF-05020182 (2) demonstrated suitable properties for further testing in vivo where it dose-dependently decreased the number of animals exhibiting full tonic extension convulsions in response to corneal stimulation in the maximal electroshock (MES) assay. In addition, PF-05020182 (2) significantly inhibited convulsions in the MES assay at doses tested, consistent with in vitro activity measure. The physiochemical properties, in vitro and in vivo activities of PF-05020182 (2) support further development as an adjunctive treatment of refractory epilepsy.

The economic burden of genetic tests for the infertile male: a pilot algorithm to improve test predictive value.

PURPOSE: We developed a model to optimize genetic testing in infertile men with nonobstructive azoospermia and severe oligospermia. We also assessed the optimal cutoff value of the predicted probability of advising genetic testing and evaluated the direct cost saving of using the model. MATERIALS AND METHODS: We retrospectively reviewed the records of infertile men who underwent Y microdeletion and karyotype testing at our fertility center from 2006 to 2012. Semen parameters, testicular volume, testosterone, luteinizing hormone, follicular stimulating hormone and varicocele were assessed as potential predictors of genetic disorders. We fitted logistic regression to all predictors and selected a nomogram based on the concordance index and calibration. We calculated the cost saving of using the model. RESULTS: Of 325 patients 278 fulfilled study inclusion criteria, including 27 with an abnormal karyotype, 11 with a Y microdeletion and 1 with each condition. We developed a nomogram using sperm concentration and motility, testicular volume and serum testosterone level. The nomogram concordance index was 0.738. The optimal cutoff value was 13.8% with 0.788 sensitivity, 0.590 specificity, 0.245 positive predictive value and 0.943 negative predictive value. Testing men above the 13.8% cutoff resulted in a direct 45% cost saving. However, 15.4% of genetic anomalies were missed, including 2 Y microdeletions. CONCLUSIONS: Using common clinical and laboratory parameters our nomogram detects 84.6% of genetic anomalies. Nomogram use resulted in a 45% direct cost saving but carries the risk of missing pertinent genetic abnormalities.

Qualitative inquiry: a method for validating patient perceptions of palliative care while enrolled on a cancer clinical trial.

BACKGROUND: Palliative care is a vital component of patient-centered care. It has increasingly become central to the management and care of seriously ill patients by integrating physical, psychosocial, and spiritual supportive services. Through qualitative inquiry, this paper examines cancer patients' perceptions of the process and outcomes of the pain and palliative care consultative services they received while enrolled in a clinical trial. METHODS: A qualitative analysis of open-ended questions was conducted from a sub-sample of patients (n = 34) with advanced cancers enrolled in a randomized controlled trial exploring the efficacy of a palliative care consult service. Two open-ended questions focused on patient perceptions of continued participation on their primary cancer clinical trials and their perceptions of interdisciplinary communication. RESULTS: THREE OVERARCHING THEMES EMERGED WHEN ASKED WHETHER RECEIVING PAIN AND PALLIATIVE CARE SERVICES MADE THEM MORE LIKELY TO REMAIN ENROLLED IN THEIR PRIMARY CANCER CLINICAL TRIAL: patients' past experiences with care, self-identified personal characteristics and reasons for participation, and the quality of the partnership. Four themes emerged related to interdisciplinary communication including: the importance of developing relationships, facilitating open communication, having quality communication, and uncertainty about communication between the cancer clinical trial and palliative care teams. CONCLUSIONS: Our findings suggest the importance of qualitative inquiry methods to explore patient perceptions regarding the efficacy of palliative care services for cancer patients enrolled in a cancer clinical trial. Validation of patient perceptions through qualitative inquiry regarding their pain and palliative care needs can provide insight into areas for future implementation research. TRIAL REGISTRATION: NIH Office of Human Subjects Research Protection OHSRP5443 and University of Pennsylvania 813365.

Global Risdiplam Compassionate Use Program for Patients with Type 1 or 2 Spinal Muscular Atrophy.

PURPOSE: Spinal muscular atrophy (SMA) is a genetic neuromuscular disease causing progressive muscle weakness and reducing life expectancy. Risdiplam (Evrysdi; Genentech/F. Hoffmann-La Roche Ltd, Basel, Switzerland) is a drug approved for use in the treatment of patients with SMA. The ongoing global risdiplam Compassionate Use Program (CUP), initiated in November 2019, is the largest CUP in SMA, currently providing access to risdiplam for >2000 patients with type 1 or 2 SMA in 59 countries. Here, the challenges and learnings from the risdiplam CUP are presented. METHODS: Enrolled patients (aged ≥2 months) had type 1 or 2 SMA and no alternative treatment options (ie, they were not medically eligible for approved SMA treatments, were unable to continue their SMA treatment due to medical reasons, were at risk for lack/loss of SMA treatment efficacy, or did not qualify for/had no access to SMA treatment within a clinical trial). Requests were made by the treating physicians via an end-to-end system. FINDINGS: The risdiplam CUP highlighted the importance of collaborating with patient advocacy groups early to learn about patients' perspectives on unmet medical needs, understanding the sometimes-unique nature of local regulations and requirements, and adapting physician- and patient-eligibility criteria. Key learnings were obtained from enrolling patients from low- to middle-income countries and from countries without dedicated Compassionate Use regulations, and from operating the CUP during the coronavirus disease 2019 pandemic. IMPLICATIONS: The risdiplam CUP experience was successful in many ways and may help to design and implement future CUPs in rare diseases, as well as patients living in countries or in circumstances in which access to innovative treatments is a challenge.

Toward quantitative super-resolution methods for cryo-CLEM.

Cryogenic ultrastructural imaging techniques such as cryo-electron tomography have produced a revolution in how the structure of biological systems is investigated by enabling the determination of structures of protein complexes immersed in a complex biological matrix within vitrified cell and model organisms. However, so far, the portfolio of successes has been mostly limited to highly abundant complexes or to structures that are relatively unambiguous and easy to identify through electron microscopy. In order to realize the full potential of this revolution, researchers would have to be able to pinpoint lower abundance species and obtain functional annotations on the state of objects of interest which would then be correlated to ultrastructural information to build a complete picture of the structure-function relationships underpinning biological processes. Fluorescence imaging at cryogenic conditions has the potential to be able to meet these demands. However, wide-field images acquired at low numeric aperture (NA) using air immersion objective have a low resolving power and cannot provide accurate enough three-dimensional (3D) localization to enable the assignment of functional annotations to individual objects of interest or target sample debulking to ensure the preservation of the structures of interest. It is therefore necessary to develop super-resolved cryo-fluorescence workflows capable of fulfilling this role and enabling new biological discoveries. In this chapter, we present the current state of development of two super-resolution cryogenic fluorescence techniques, superSIL-STORM and astigmatism-based 3D STORM, show their application to a variety of biological systems and discuss their advantages and limitations. We further discuss the future applicability to cryo-CLEM workflows though examples of practical application to the study of membrane protein complexes both in mammalian cells and in Escherichia coli.

Rehabilitation via HOMe-Based gaming exercise for the Upper limb post Stroke (RHOMBUS): a qualitative analysis of participants' experience.

OBJECTIVE: To report participants' experiences of trial processes and use of the Neurofenix platform for home-based rehabilitation following stroke. The platform, consisting of the NeuroBall device and Neurofenix app, is a non-immersive virtual reality tool to facilitate upper limb rehabilitation following stroke. The platform has recently been evaluated and demonstrated to be safe and effective through a non-randomised feasibility trial (RHOMBUS). DESIGN: Qualitative approach using semistructured interviews. Interviews were audio recorded, transcribed verbatim and analysed using the framework method. SETTING: Participants' homes, South-East England. PARTICIPANTS: Purposeful sample of 18 adults (≥18 years), minimum 12 weeks following stroke, not receiving upper limb rehabilitation prior to the RHOMBUS trial, scoring 9-25 on the Motricity Index (elbow and shoulder), with sufficient cognitive and communicative abilities to participate. RESULTS: Five themes were developed which explored both trial processes and experiences of using the platform. Factors that influenced participant's decision to take part in the trial, their perceptions of support provided during the trial and communication with the research team were found to be important contextual factors effecting participants' overall experience. Specific themes around usability and comfort of the NeuroBall device, factors motivating persistence and perceived effectiveness of the intervention were highlighted as being central to the usability and acceptability of the platform. CONCLUSION: This study demonstrated the overall acceptability of the platform and identified areas for enhancement which have since been implemented by Neurofenix. The findings add to the developing literature on the interface between virtual reality systems and user experience. TRIAL REGISTRATION NUMBER: ISRCTN60291412.

Microenvironment shapes small-cell lung cancer neuroendocrine states and presents therapeutic opportunities.

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intratumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the cell-extrinsic drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the TME exhibit substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAFs) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLC's adaptable nature, opening possibilities for reprogramming the TME-tumor communications that shape SCLC tumor states.

The nature of life-transforming changes among cancer survivors.

Some cancer survivors report positive subjective changes they describe as "life transforming." We used a grounded theory approach to identify the content, underlying process, and identifying characteristics of self-defined "life-transforming" changes (LTCs) reported by 9 cancer survivors. To actualize their hopes for improvement, participants used a self-guided process centered on pragmatic action: researching options, gaining experience, and frankly evaluating results. Many participants discovered unanticipated personal abilities and resources, and those became highly useful in coping with other challenges apart from cancer. This made the increased personal abilities and resources "life transforming" rather than being substantially limited to reducing cancer-related problems. The action-oriented features and processes of LTCs seemed to be more fully described by experiential learning theory than by posttraumatic growth and coping. Supportive intervention to facilitate positive change processes could decrease suffering and enhance positive psychosocial and spiritual outcomes for cancer survivors.

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli.

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage.

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5-14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54-1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.