Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

The Epidemiology of Injuries in Adults in Nepal: Findings from a Hospital-Based Injury Surveillance Study.

This study aimed to develop and evaluate a model of hospital-based injury surveillance and describe the epidemiology of injuries in adults. One-year prospective surveillance was conducted in two hospitals in Hetauda, Nepal. Data were collected electronically for patients presenting to emergency departments (EDs) with injuries between April 2019 and March 2020. To evaluate the model's sustainability, clinical leaders, senior managers, data collectors, and study coordinators were interviewed. The total number of patients with injuries over one year was 10,154, representing 30.7% of all patients visiting the EDs. Of patients with injuries, 7458 (73.4%) were adults aged 18 years and over. Most injuries (6434, 86%) were unintentional, with smaller proportions due to assault (616, 8.2%) and self-harm (408, 5.5%). The median age of adult patients was 33 years (IQR 25-47). Males had twice the rate of ED presentation compared with females (40.4 vs. 20.9/1000). The most common causes were road traffic accidents (32.8%), falls (25.4%), and animal/insect related injuries (20.1%). Most injured patients were discharged after treatment (80%) with 9.1% admitted to hospital, 8.1% transferred to other hospitals, and 2.1% died. In Nepal, hospital-based injury surveillance is feasible, and rich injury data can be obtained by embedding data collectors in EDs.

Development and Evaluation of a Community Surveillance Method for Estimating Deaths Due to Injuries in Rural Nepal.

Almost 10% of global deaths are secondary to injuries, yet in the absence of routine injury surveillance and with few studies of injury mortality, the number and cause of injury deaths in many countries are not well understood. This study aimed to develop and evaluate the feasibility of a method to identify injury deaths in rural Nepal. Working with local government authorities, health post staff and female community health volunteers (FCHVs), we developed a two-stage community fatal injury surveillance approach. In stage one, all deaths from any cause were identified. In stage two, an interview with a relative or friend gathered information about the deceased and the injury event. The feasibility of the method was evaluated prospectively between February 2019 and January 2020 in two rural communities in Makwanpur district. The data collection tools were developed and evaluated with 108 FCHVs, 23 health post staff and two data collectors. Of 457 deaths notified over one year, 67 (14.7%) fatal injury events were identified, and interviews completed. Our method suggests that it is feasible to collect data on trauma-related deaths from rural areas in Nepal. These data may allow the development of injury prevention interventions and policy.

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions.

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity - due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra -, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.

Perceptions of injury risk in the home and workplace in Nepal: a qualitative study.

OBJECTIVE: Injuries are a global health problem. To develop context-specific injury prevention interventions, one needs to understand population perceptions of home and workplace injuries. This study explored a range of views and perceptions about injuries in a variety of settings and identified barriers and facilitators to injury prevention. DESIGN: Qualitative study: interviews and focus groups. SETTING: Three administrative areas: Hetauda submetropolitan city, Thaha municipality and Bakaiya rural municipality in Makwanpur, Nepal. PARTICIPANTS: Nine focus groups (74 participants) and nine one-to-one interviews were completed; workers from diverse occupations, residents (slum, traditional or modern homes) and local government decision-makers participated in the study between May and August 2019. The interviews and discussions were audio-recorded, transcribed verbatim, translated to English and analysed thematically. RESULTS: Six themes were developed: unsafe home and workplace environment; inadequate supervision and monitoring; perceptions that injuries are inevitable; safety takes low priority: financial and behavioural considerations; safety education and training; and government-led safety programmes and enforcement. Key barriers to injury prevention were perceived to be lack of knowledge about injury risk and preventive measures both at the community level and at the workplace. Facilitators were community-level educational programmes and health and safety training to employees and employers. Participants stressed the importance of the role of the government in planning future injury prevention programmes in different environments. CONCLUSIONS: This study highlighted that both home and workplace injuries are complex and multifactorial. Lack of knowledge about injury risks and preventive measures, both at the community level and at the workplace, was found to be a common barrier to injury prevention, perceived to be mitigated by educational programmes. Together with previously published epidemiological evidence, the barriers and facilitators identified in this study offer useful basis to inform policy and practice.

Home-related and work-related injuries in Makwanpur district, Nepal: a household survey.

OBJECTIVE: To describe the epidemiology of home-related and work-related injuries, their mechanisms, inequalities and costs associated with these injuries. METHODS: A household survey was undertaken in three palikas of Makwanpur district between April and June 2019. Data were collected electronically on non-fatal injuries that occurred in the previous 3 months and fatal injuries that occurred in the previous 5 years. FINDINGS: 17 593 individuals were surveyed from 3327 households. Injury rates were 8.0 per 1000 population for home injuries and 6.4 per 1000 for work-related injuries; 61.0% of home injuries were among women and 69.9% of work-related injuries among men. Falls were the cause of 48% home injuries, affecting 50.9% of men and 46.5% of women. Burns/scalds were higher in women than men, affecting 17.4% of women reporting home injuries. Cuts and piercings accounted for 39.8% of all work-related injuries and 36.3% were falls. Injury incidence varied by ethnic group: home injuries were highest in Brahmin (12.0 per 1000) and work-related injuries highest in Rai groups (21.0 per 1000). The total mean costs (transport and treatment) of work-related injury was US$143.3 (SD 276.7), higher than for home injuries (US$130.4, SD 347.6). The number of home (n=74, 64.9%) and work-related (n=67, 77.9%) injuries were higher in families below the poverty line than families in the next income bracket (home: n=22, 19.3%; work: n=11, 12.8%). CONCLUSIONS: Home-related and work-related fall injuries are common. The inequalities in injury identified in our study by rurality, age, sex, income level and ethnic group can help target injury prevention interventions for vulnerable groups.

Long-term impact of community-based participatory women's groups on child and maternal mortality and child disability: follow-up of a cluster randomised trial in rural Nepal.

BACKGROUND: Community-based women's groups practising participatory learning and action (PLA) can reduce maternal and neonatal mortality in low-income countries. However, it is not clear whether these reductions are associated with subsequent increased or decreased rates of childhood death and disability. We assessed the impact on child deaths and disability beyond the perinatal period among participants in the earliest trial in Nepal 2001-2003. METHODS: Household interviews were conducted with mothers or household heads. At cluster and individual levels, we analysed disability using pairwise log relative risks and survival using multilevel logistic models. FINDINGS: From 6075 children and 6117 mothers alive at 4 weeks post partum, 44 419 children (73%) were available for interview a mean 11.5 years later. Rates of child deaths beyond the perinatal period were 36.6 and 52.0 per 1000 children in the intervention and control arms respectively. Rates of disability were 62.7 and 85.5 per 1000 children in the intervention and control arms respectively. Individual-level analysis, including random effects for cluster pairing and adjusted for baseline maternal literacy, socioeconomic status and maternal age, showed lower, statistically non-significant, odds of child deaths (OR 0.70 (95% CI 0.43 to 1.18) and disability (0.64 (0.39 to 1.06)) in the intervention arm. CONCLUSION: Community-level exposure to women's groups practising PLA did not significantly impact childhood death or disability or death beyond the perinatal period. Follow-up of other trials with larger sample sizes is warranted in order to explore the possibility of potential long-term survival and disability benefits with greater precision.

Prenatal and perinatal risk factors for disability in a rural Nepali birth cohort.

BACKGROUND: Improving newborn health remains a global health priority. Little however is known about the neurodevelopmental consequences for survivors of complications in pregnancy, labour and the neonatal period in in low-income countries outside of small selective and typically urban facility studies. We ask which antenatal, birth and neonatal factors are associated with disability in childhood in a large community birth cohort from rural Nepal. METHODS: 6436 infants were recruited during a cluster randomised control trial (RCT) of participatory women's groups (ISRCTN31137309), of whom 6075 survived beyond 28 days. At mean age of 11∙5 years (range 9.5-13.1), 4219 children (27% lost to follow-up) were available for disability screening which was conducted by face-to-face interview using the Module on Child Functioning and Disability produced by the Washington Group/UNICEF. Hypothesised risk factors for disability underwent multivariable regression modelling. FINDINGS: Overall prevalence of disability was 7.4%. Maternal underweight (OR 1.44 (95% CI 1.01-2.08)), maternal cohabitation under 16 years of age (OR 1.50 (1.13-2.00)), standardised infant weight at 1 month (OR 0.82 (0.71-0.95)) and reported infant diarrhoea and vomiting in the first month (OR 2.48 (1.58-3.89)) were significantly associated with disability adjusted for trial allocation. The majority of hypothesised risk factors, including prematurity, were not significant. INTERPRETATION: Proxies for early marriage and low birth weight and a measure of maternal undernutrition were associated with increased odds of disability. The lack of association of most other recognised risk factors for adverse outcome and disability may be due to survival bias.

Disabled women׳s maternal and newborn health care in rural Nepal: a qualitative study.

OBJECTIVE: there is little evidence about disabled women׳s access to maternal and newborn health services in low-income countries and few studies consult disabled women themselves to understand their experience of care and care seeking. Our study explores disabled women׳s experiences of maternal and newborn care in rural Nepal. DESIGN: we used a qualitative methodology, using semi-structured interviews. SETTING: rural Makwanpur District of central Nepal. PARTICIPANTS: we purposively sampled married women with different impairments who had delivered a baby in the past 10 years from different topographical areas of the district. We also interviewed maternal health workers. We compared our findings with a recent qualitative study of non-disabled women in the same district to explore the differences between disabled and non-disabled women. FINDINGS: married disabled women considered pregnancy and childbirth to be normal and preferred to deliver at home. Issues of quality, cost and lack of family support were as pertinent for disabled women as they were for their non-disabled peers. Health workers felt unprepared to meet the maternal health needs of disabled women. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: integration of disability into existing Skilled Birth Attendant training curricula may improve maternal health care for disabled women. There is a need to monitor progress of interventions that encourage institutional delivery through the use of disaggregated data, to check that disabled women are benefiting equally in efforts to improve access to maternal health care.