Tuberculosis (TB) remains a significant healthcare problem. Understanding physical and functional impairments that patients with active TB present with at the time of diagnosis and how these impairments change over time while they receive anti-TB therapy is important in developing appropriate rehabilitation programmes to optimise patients’ recovery.The aim of this study was to assess the acceptability, implementation and practicality of conducting a prospective, observational and longitudinal trial to describe physical and functional impairments of patients with active TB.A feasibility pilot study was performed. Patients with acute pulmonary TB admitted to an urban quaternary-level hospital were recruited. Physical  and functional (exercise capacity) outcomes were assessed in hospital, and at 6 weeks and 6 months post-discharge. Descriptive statistics were used to analyse the data.n = 5; 41.7%) and mortality  rates were observed. Limitations identified regarding study feasibility included participant recruitment rate, equipment availability and suitability of outcome measures. Participants’ mean age was 31.5 (9.1) years and the majority were human immunodeficiency virus (HIV) positive . Non-significant changes in muscle architecture and power were observed over 6 months. Balance impairment was highlighted when vision was removed during testing. Some improvements in 6-minute walk test distance were observed between hospitalisation and 6 months.High dropout (Success of a longitudinal observational trial is dependent on securing adequate funding to address limitations observed related to equipment availability, staffing levels, participant recruitment from additional study sites and participant follow-up at community level. Participants’ physical and functional recovery during anti-TB therapy seems to be limited by neuromusculoskeletal factors. Tuberculosis (TB) remains a very significant and pertinent healthcare problem. It is currently the leading cause of mortality associated with a single infectious pathogen globally, and one of the top 10 causes of death worldwide WHO . In 2016sequelae [a condition which is the consequence of a previous disease or injury] that survivors of acute illness may experience, such as impairments in functional status and quality of life (QOL)  at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) with active TB infection were considered for participation in this project. Charlotte Maxeke Johannesburg Academic Hospital is an accredited central university-affiliated quaternary-level government hospital in Johannesburg that offers a range of specialist inpatient and outpatient services to residents across Gauteng and neighbouring provinces. Diagnosis of TB was made through patient clinical presentation, radiological investigations and on-site access to GeneXpert for confirmation of pulmonary TB. A sputum sample was sent for GeneXpert for all patients with suspected TB and results were obtained on the day of testing. Urine lipoarabinomannan (LAM) testing and tissue biopsy were performed for select patients when deemed appropriate to assist with diagnosis. Physiotherapy service provision at CMJAH involved inpatient management only and no post-discharge physiotherapy services were offered to patients with TB without neurological symptoms. Post-discharge follow-up of participants was done at the Physiotherapy Department Movement Laboratory on the University of the Witwatersrand Education Campus in Parktown, which is situated opposite CMJAH.A consecutive sampling method was used to recruit participants for this feasibility pilot study. Both male and female patients 18 years or older, with or without HIV seropositivity, who were diagnosed with active TB infection were approached regarding possible participation. Subjects were not considered for participation if they had TB meningitis, were unable to stand and walk without assistance, had recent neurosurgery or head injury with loss of consciousness for more than 10 min, recent orthopaedic injury to the lower limbs, amputation, diagnosed major comorbidity , ocular disease or vestibular disorders.The study was conducted over a 15-month period and all eligible patients who provided consent were included. Participant recruitment for baseline hospital assessment was performed over a 12-month period (May 2016–May 2017). Six-month follow-up assessments for all participants were completed by August 2017.Two of the researchers (H.v.A. and S.Q.) performed testing on all participants. Prior to the pilot study these researchers underwent training in the use of all of the instrumentation listed below.The Berg balance scale was used to assess participants’ balance. It is a 14-item objective measure that assesses static and dynamic balance and risk for falls in adults. Item-level scores range from 0 to 4, and scores for each item are added together to determine each participant’s score achieved out of a maximum score of 56. It is a reliable and valid tool for the assessment of balance in a variety of populations, including those with respiratory diseases  for 10 s, and single-leg squat. These tests were added as it was anticipated that there might be a ceiling effect of the Berg balance scale scores in this patient population.The timed up-and-go (TUG) test was used to assess functional balance. It assesses a participant’s ability to stand up from a chair, walk 3 m, turn around, walk back to the chair and sit down. It also assesses the time in which these activities are completed. Duration of greater than 13.5 s predicts a high risk of falls. The TUG test is a widely used reliable and valid tool for the assessment of functional balance  was used to determine participants’ exercise capacity. It is a functional walk test that assesses the distance a person can walk on a flat surface in 6 min using a 30-m track. Instructions on performance of the 6MWT, as outlined by the American Thoracic Society guidelines , were imLevel of dyspnoea was assessed using the modified Borg (0–10) scale. The higher the score, the more limited a participant’s function would be as a result of dyspnoea. It is a reliable and valid tool for the measurement of level of dyspnoea in patients with respiratory diseases  in the transverse image plane using the on-screen calliper to manually outline the inner echogenic line that represented the fascia around the rectus femoris muscle , thickness (mass) and echo intensity. A DP-6600 model digital ultrasonic diagnostic imaging system  with a 5-MHz curvilinear transducer and a large footprint (≥60 mm) was used. B-mode was selected to image each participant’s rectus femoris and vastus lateralis muscles of their dominant leg. These muscles form part of the quadriceps muscle group and play an important role in facilitating hip flexion and knee extension actions, while a person performs physical activities such as walking, running and jumping. Measurements were done with on-screen callipers. Muscle CSA was measured  was used to assess muscle power. The power of three predetermined upper extremity and three predetermined lower extremity muscle groups is assessed bilaterally on a scale of 0–5, where 0 represents no visible muscle contraction and 5 represents normal muscle power (against gravity and resistance). The maximum score that can be achieved is 60 with normal muscle power reflected by a score closer to 60/60. The MRC-SS is a widely used reliable and valid tool to assess muscle power in various patient populations, including patients with acute illness . Potential participants were identified by the nurse in charge of the unit. The researcher (M.M.C.), in the presence of the charge nurse, approached these potential participants to screen them for eligibility against the inclusion and exclusion criteria as soon as their condition had stabilised. Detailed explanation of all tests and procedures involved were provided to participants. They were given 24 h to consider participation before written consent was obtained (by M.M.C.). Demographic information  was obtained from the medical file of each consenting participant. A short interview was held (by M.M.C.) with each participant, while they were seated comfortably in bed or in a chair, to obtain additional information . The information collected from each participant was used as a baseline assessment.At the start of the assessment, each participants’ level of dyspnoea was assessed with the modified Borg scale and recorded. For ultrasound imaging, participants were placed in a supine position on their hospital bed with both knees extended but relaxed and toes pointing to the ceiling . The scanning site for the rectus femoris muscle was determined by identifying the mid-point on the line from the greater trochanter of the femur to the knee joint line. A generous amount of ultrasound contact gel was placed on the skin surface over the scanning site before the transducer was placed over this site applying minimal pressure. The transducer was placed perpendicular to the femur to visualise the rectus femoris muscle and obtain baseline measurements for muscle CSA and thickness. The transducer was placed parallel to the femur to visualise the vastus lateralis muscle to obtain baseline measurements for echo intensity. Two measurements for each outcome were taken and the average of each of these measurements was recorded Pillen .Muscle power was assessed with participants sitting upright over the edge of the hospital bed. The MRC-SS was used to assess power of shoulder abduction, elbow flexion and wrist extension movements of bilateral upper extremities. Hip flexion, knee extension and ankle dorsiflexion power were assessed for both the right and left lower extremities. Because the MRC-SS involves volitional muscle testing, strong verbal encouragement was given to each participant during the test  before their balance was assessed with the Berg balance scale and the two additional balance tests. This involved participants performing various predetermined activities in sitting and standing by the bedside to assess static and dynamic balance and risk of falls. Next, functional balance was assessed with the TUG test. Participants were asked to sit in an upright chair by their bedside. A stopwatch was used to measure the total duration for participants to stand up from the chair, cover the 3-m distance, turn around, return to the chair and sit down. The time to completion of the test was recorded and a note was made of any assistive devices that the participant used during the test.2) and recorded. Thereafter the test was commenced. Participants were instructed to walk at a comfortable pace of moderate intensity to ensure as much distance as possible is covered and that an adequate exercise response was obtained during the test. Encouragement to participants during the walk test was given strictly according to the prescribed guidelines  verified each participant’s details and demographic information with them and changes in details were recorded. All outcome measures assessed during hospitalisation were repeated at both of these assessment points.The data obtained were nominal, ordinal and ratio in nature. Data analysis was performed using IBM® SPSS version 24 for Windows software and intention-to-treat analysis was done. Missing data for those who were still alive but dropped out of the trial were managed through last observation carried forward. Descriptive statistics were used to present the data and normality of data was tested using the Shapiro–Wilk test. Categorical data were summarised as frequencies and percentages. Continuous data were presented as mean, and standard deviations (SD) for normally distributed data and median and interquartile range (IQR) for non-normally distributed data. To determine changes in outcomes over time, ANOVA with repeated measures with a Greenhouse–Geisser correction was performed using Bonferroni confidence interval adjustment. The level of significance was set at ≤0.05 with a 95% confidence interval (CI). No regression analysis was performed because of the small sample size.Permission to conduct the study was obtained from the University of the Witwatersrand Human Research Ethics  committee (certificate number: M150857) and the hospital review board. The study was registered with the South African National Clinical Trials Registry . The principles for conducting research on human subjects, as outlined in the Declaration of Helsinki, were adhered to throughout the duration of the trial.n = 5) was observed over the 6 months of the observation period.Sixteen participants were enrolled into the study during the 12 months of the recruitment period. n = 12) was 31.5 (9.1) years. Reported symptoms experienced before admission varied widely and included shortness of breath, vomiting, headaches, feeling disoriented, tiredness and night sweats. Only two participants were admitted to ICU and had a mean duration of stay of 1.1 (2.3) days. One was lost to follow-up after hospital discharge and the other died before the 6 weeks follow-up assessment. Participants were commenced on anti-TB therapy on average within 2 days from admission. Most were managed with first-line anti-TB therapy (Rifafour and Ethambutol), but two presented with drug-resistant TB and were managed with Ethionamide, Isoniazid and Pyrazinamide. The median duration of hospitalisation for the whole group (n = 12) was 6.5 (IQR 4.8–16.5) days. n = 10, 83.3%) and most participants  presented with pulmonary TB and were HIV seropositive . The majority of participants  did not partake in exercise prior to admission and none of the participants reported a history of smoking. Few participants had returned to employment at 6 months.The mean age of participants (2) occurred between the 6 weeks and 6 months of assessments, whereas the greatest changes in mean muscle thickness (2.3 mm) and mean echo intensity (5.2%) occurred between the hospital assessment and 6 weeks follow-up. Changes observed for these parameters over the 6-month period were, however, not significant .Findings for muscle CSA, thickness and echo intensity are summarised in n = 10) was 51.2 , which changed to 52  at 6 weeks (n = 7), with a mean MRC-SS of 52.8  at 6 months post-discharge (n = 6). A mean change in muscle power of 0.8 was observed between hospital assessment and 6 weeks follow-up and between 6 weeks and 6 months post-discharge, respectively. The changes observed in MRC-SS over the 6-month period were not significant (p = 0.3).The mean MRC-SS during hospitalisation for this cohort (n = 10), 54.5  and 54.7 , respectively, at the three assessment points. Four of the 10 participants (40%) managed to achieve the highest score (56/56) at hospital assessment. The greatest change in mean scores (0.8) was observed between the hospital assessment and 6 weeks follow-up. The changes observed in mean Berg scores over the 6-month period were not significant (p = 0.18).Balance was assessed with the Berg balance scale and the TUG test. The mean Berg balance scores were 53.7  s. At 6 weeks they completed the test in a mean duration of 9.5  s and at 6 months in 9.8  s. None of the participants used an assistive device during the TUG test assessment at any of the three assessment points. A high risk of falls was identified in only one participant during hospitalisation and in no participants at 6 weeks or 6 months after discharge. The greatest change in mean TUG test duration (0.7 s) was observed between hospital assessment and 6 weeks. The changes observed in TUG test duration over the 6-month period were not significant (p = 0.36).During hospitalisation, participants completed the TUG test in a mean duration of 10.2  was 1.5 , which represented ‘very slight’ and ‘slight’ breathlessness. The mean score changed to 1  at 6 weeks (n = 7), and 1.1  at 6 months (n = 6) post-discharge, which represents ‘very slight’ breathlessness. The greatest change in mean Borg scores (0.5) was between hospitalisation and the 6 weeks follow-up assessment. The changes observed in modified Borg scores over the 6-month period were not significant (p = 0.31).The mean Borg score during hospitalisation . There was reduction in mean peripheral oxygen saturation levels between rest and immediately after completion of the 6MWT of −0.14% at hospital assessment. This changed to −3.1% at 6 weeks and −2% at 6 months. After 5-min rest, there was an improvement in mean peripheral oxygen saturation levels of 4.3%, 3.7% and 3% at the three assessment points, respectively.The 6MWT was performed over a 10-m distance on the hospital ward because of lack of open space available. At 6 weeks and 6 months, a 30-m track was used in the Physiotherapy Department as recommended by the American Thoracic Society  guidelinThis study was conducted to assess the acceptability, implementation and practicality of conducting a prospective, observational and longitudinal trial in an urban quaternary-level hospital setting to evaluate physical and functional impairments of patients diagnosed with active TB infection. In addition, preliminary findings on changes in muscle architecture, mass and power, level of dyspnoea, balance and exercise capacity over the study period are presented.n = 5, 41.7%) was, however, observed over the 6 months follow-up period. Attempts made by the researchers to prevent loss to follow-up such as written information given to participants regarding appointment dates and times and venue prior to discharge, and reminder telephone calls and text messaging seemed to be unsuccessful in some cases. Some participants provided contact details during the hospital assessment session that proved to be incorrect after they were discharged as their mobile phone numbers were not in service. In some cases the calls made went unanswered. During telephonic conversation, some participants who dropped out of the study after hospital discharge commented that they did not have the financial means to return to the test venue for follow-up assessments. This was stated despite the fact that funding was in place to reimburse participants for their travel costs from home to the Wits University Education Campus for follow-up assessment. This information was verbalised to them prior to hospital discharge. Others stated that they felt too weak to endure the travel journey from their homes to the test venue.Acceptability in feasibility studies refers to how participants in a trial react to an intervention . Participants in this study were keen to perform the tests used to assess their levels of physical and functional impairments while in hospital. No adverse events occurred as a result of any of the test procedures. A high dropout rate . A large portion of participants achieved the highest score on the Berg balance scale during initial hospital assessment. An instrument presents with a ceiling effect if more than 15% of participants achieve the highest possible score . This suggests that the Berg balance scale might not be sensitive enough to accurately detect balance disturbances in this patient population; however, the sample size for this study was small and therefore findings should be interpreted with caution. Alternatively, the Y-balance test might be more appropriate for this patient population as it assesses dynamic balance. Its reliability and effectiveness has been demonstrated in middle-aged and older healthy females . During the 12 months of recruitment period, of the 26 patients screened, only 16 patients agreed to participate in the study. The rate of recruitment from this single study site was slower than initially anticipated. Consideration should, thus, be given to involving more hospital sites from which potential participants can be recruited for a longitudinal study. The relatively high post-discharge mortality rate observed during this feasibility study (33.3%) was concerning as it creates challenges for participant retention when using a longitudinal study design. This would be another motivating factor for involving more than one study site for longitudinal trials. Limited availability of the ultrasound imaging equipment was problematic and therefore purchase of one additional device is recommended.The young cohort in this study presented with small non-significant improvements in muscle power over the 6-month observation period. Skeletal muscle power is dependent on skeletal muscle mass, composition and architecture   more than by respiratory symptoms particularly between 6 weeks and 6 months of anti-TB therapy.The abnormalities detected in rectus femoris and vastus lateralis muscle composition and limitations observed in general muscle power, complex balance activities and exercise capacity in this small cohort of patients on anti-TB therapy motivate for further exploration in longitudinal trials, provided that adequate funding can be secured. Findings from such longitudinal trials can be used to lobby for extension of rehabilitation services provided to this patient population, beyond hospital discharge, to optimise their physical and functional recovery at completion of anti-TB therapy.


Depression is underdiagnosed and undertreated in primary health care. When associated with chronic physical disorders, it worsens outcomes. There is a clear gap in the treatment of depression in low- and middle-income countries (LMICs), where specialists and funds are scarce. Interventions supported by mobile health  technologies may help to reduce this gap. Mobile phones are widely used in LMICs, offering potentially feasible and affordable alternatives for the management of depression among individuals with chronic disorders.This study aimed to explore the potential effectiveness of an mHealth intervention to help people with depressive symptoms and comorbid hypertension or diabetes and explore the feasibility of conducting large randomized controlled trials (RCTs).Emotional Control (CONEMO) is a low-intensity psychoeducational 6-week intervention delivered via mobile phones and assisted by a nurse for reducing depressive symptoms among individuals with diabetes or hypertension. CONEMO was tested in 3 pilot studies, 1 in São Paulo, Brazil, and 2 in Lima, Peru. Depressive symptoms were assessed using the Patient Health Questionnaire-9 (PHQ-9) at enrollment and at 6-week follow-up.The 3 pilot studies included a total of 66 people. Most participants were females aged between 41 and 60 years. There was a reduction in depressive symptoms as measured by PHQ-9 in all pilot studies. In total, 58% (38/66) of the participants reached treatment success rate (PHQ-9 <10), with 62% (13/21) from São Paulo, 62% (13/21) from the first Lima pilot, and 50% (12/24) from the second Lima pilot study. The intervention, the app, and the support offered by the nurse and nurse assistants were well received by participants in both settings.The intervention was feasible in both settings. Clinical data suggested that CONEMO may help in decreasing participants’ depressive symptoms. The findings also indicated that it was possible to conduct RCTs in these settings. Depression is a leading cause for the burden of disease worldwide, with 4.4% of the world’s population suffering from depression ,2. DepreIn LMICs, people with depression are commonly underdiagnosed and undertreated . One in Emotional Control (CONEMO), a low-intensity psychoeducational 6-week intervention delivered via mobile phones assisted by a nurse for reducing depressive symptoms among individuals with diabetes or hypertension.The Latin America Treatment and Innovation Network in Mental Health seeks to develop and test a blended intervention for depression and comorbid chronic health problems such as hypertension and/or diabetes. We developed The intervention aims to reduce depressive symptoms among patients with comorbid diabetes or hypertension recruited from different health service units in São Paulo (Brazil) and Lima (Peru). Therefore, the focus of this project is on providing patients tools through mobile phones, with low-intensity support that can be provided in minimal time by nurses or nurse assistants (NAs), who are available in the clinics but have no specialized training in mental health. These professionals receive specialized supervision provided by the study staff to perform their tasks. This intervention is unique in LMICs in terms of using a technological platform and integrating the care of chronic mental and physical conditions .This pilot study aimed to:Explore the potential effectiveness and feasibility of using the CONEMO intervention to help people with or hypertension or diabetes and comorbid depressive symptoms;Test recruitment strategies to estimate how many participants should be screened to reach our target sample for fully powered randomized controlled trials (RCTs);Assess acceptability and satisfaction of patients with the CONEMO intervention.We conducted 3 pilot studies between 2015 and 2016, of which 1 was in São Paulo, Brazil, and 2 in Lima, Peru. São Paulo is the largest city of Brazil with 11.2 million inhabitants . Brazil Lima, the capital of Peru, has approximately 9 million inhabitants, one-third of the total Peruvian population . The preIn São Paulo we conducted the pilot study with 4 teams from 2 FHCS clinics. In Lima, the first pilot study took place in the endocrinology and cardiology outpatient consultation area of a tertiary-level hospital from the MINSA, whereas in the second pilot, patients were recruited in 2 primary care health centers, mostly in the elderly adult consultation programs, where people with noncommunicable diseases are monitored, at EsSalud.Participants were eligible according to the following inclusion criteria:Aged 21 years or above;Had hypertension or diabetes;Had depressive symptoms, evaluated by the PHQ-9 (score ≥10); andSelf-reported reading ability in Portuguese (São Paulo) or Spanish (Lima).The exclusion criteria were as follows:Psychosis symptoms detected by a 5-item screening questionnaire;Pregnancy (self-reported);High suicidal risk detected by a positive answer in item 9 of the PHQ-9 followed by a protocol to assess suicidal risk (Suicide Risk Assessment Protocol); andCognitive impairment detected by a 4-item questionnaire.We used convenience samples in the 3 pilot studies. We envisaged that with 20 participants in each pilot, we would be able to achieve the aims proposed.We collected information about socioeconomic and demographic characteristics, depressive symptoms, suicidal ideation, presence of psychosis symptoms, cognitive impairment, quality of life, adherence to medical treatment, and disabilities .To explore the potential effectiveness of the CONEMO intervention, we used the Patient Health Questionnaire-9 (PHQ-9) as the main measure of outcome . We applTo rule out the presence of psychotic symptoms, we used the Psychosis Screening Questionnaire ; to assedisagree and 5 is agree, at the end of the pilot study.We also used data from participants captured automatically by the CONEMO system to monitor patients’ progress, including completion of sessions, intervals between accessing sessions, or sessions missed. We collected information from participants about the acceptability and satisfaction with the CONEMO app and the dashboard through a questionnaire using a Likert scale of 5 points, where 1 is We tested an intervention that could be delivered in existing general medical care settings by available local personnel. The choice of a psychological intervention as the main component relates to the public health need for these approaches in most LMICs. CONEMO is a mobile phone-delivered intervention, supported by an NA or a nurse, using a task-shifting model, as recommended by the World Health Organization ,30. CONEThe tasks of the nurse or NA were as follows: (1) an initial contact by phone to schedule a training session for the participant; (2) a face-to-face training session to introduce the CONEMO app; (3) a monitoring call after week 1 to answer possible queries from participants and to reinforce motivation to participate; (4) a monitoring call after week 4 with the same objectives as above; (5) a phone call to schedule the closing meeting; and (6) a final face-to-face appointment with the participant to finish the intervention and collect the mobile phone. Nurses or NAs received weekly face-to-face supervision from research clinical psychologists.To test recruitment strategies, we analyzed the data gathered during the process of recruiting and enrolling participants (Aim 2). The comparison of participants’ PHQ-9 scores at baseline and at follow-up allowed a preliminary assessment of potential effectiveness (Aim 1), and the number of sessions completed and ratings on the satisfaction with the CONEMO intervention gave us an insight into the acceptance of the CONEMO intervention (Aim 3).The main outcome measure was patients’ PHQ-9 scores at the end of the 6-week intervention. We considered that patients had recovered when the PHQ-9 score at follow-up was <10. The secondary outcomes, such as disability levels and quality of life, were also examined at the end of the pilots. Descriptive statistics were also used to assess participants’ perceptions at the end of the study. We conducted all data analysis using STATA 11 by StataCorp .The pilot studies were approved by local Institutional Review Boards and the US National Institute of Mental Health Data and Safety Monitoring Board. Participants consented to participate in the study and interviews took place after collecting the participants’ signatures on informed consent forms. All investigators and research assistants involved in the data collection and analysis completed ethics and human research good clinical practices training before starting their activities in the project.We carried out 1 pilot study in São Paulo and 2 pilot studies in Lima. In São Paulo, 4 nurses from the 2 participating FHCS clinics were responsible to support the CONEMO intervention for participants recruited into the study. In the first pilot in Lima, we employed a nurse to support the delivery of the intervention, whereas in the second pilot study in Lima, we worked with 6 nurses employed by 2 public health care facilities. We included 66 patients in the 3 pilot studies after approaching 793 people . Of the At the 6-week follow up, there was 5% (1/21%) loss to follow-up in São Paulo and 28.6% (6/21) in the first and 33% (8/24) in the second Lima pilot studies. Among participants who completed follow-up assessments, 11 out of 20 (55%) had moderately severe or severe symptoms of depression at baseline in São Paulo . In LimaIn São Paulo and Lima, all participants presented some level of functional disability at baseline, with more than 50% presenting moderate or severe functional disability. In all pilot studies, there was a trend toward a reduction in levels of functional disability at the follow-up assessments . RegardiAccess of participants to the CONEMO sessions decreased progressively over time in the São Paulo and in the second Lima pilot study , whereasThe CONEMO app (participant interface) worked adequately during the trial, despite some connectivity issues when receiving information on participants’ performance. These issues did not affect the intervention or data collection.We collected data on acceptability and satisfaction with CONEMO from 20 participants in São Paulo, 15 in Lima’s first pilot, and 16 in Lima’s second pilot. The evaluation of the intervention by participants was generally positive: all mean ratings were above 3.5 points (maximum of 5 points), both in São Paulo and Lima. Patients considered that the objectives were attained and that the intervention helped their physical and mental health and helped them to get organized. In their opinion the duration of the intervention, the amount of contacts with the nurse or NA, and the training received were adequate .Participants considered the CONEMO app easy to use, were able to access videos and other resources included in the app, and thought the size of fonts and layout of the app was appropriate . They alThe main aim of this pilot study was to explore the feasibility and potential effectiveness of the CONEMO intervention for depressed people with comorbid hypertension or diabetes. We also wanted to assess the feasibility of undertaking fully powered RCTs to test the effectiveness of this intervention. We found that patients were able to use the CONEMO app, that they tended to show improvement in the severity of depressive symptoms by the end of the intervention, and that they were satisfied with the intervention. Our results are encouraging and suggest that the intervention is feasible and potentially effective with this population.To carry out RCTs, we needed to ensure that we would be able to recruit participants in the numbers needed. On average, out of every 13 participants screened, we were able to include 1 eligible participant in the study. In São Paulo and Lima, recruitment took place in primary health care units and, additionally, in the outpatient consultation areas of a public hospital in the first pilot study in Lima, with patients presenting different conditions apart from hypertension and diabetes. In both sites, many participants were unable to read or write. As a result, it was necessary to prescreen twice as many patients than we first anticipated to reach the numbers we had planned for each pilot study. This information allowed us to prepare a more realistic recruitment strategy and plan.The instruments and measures were well understood by participants, and research assistants also provided positive feedback on their use. The outcome measures seemed sensitive to change and allowed a useful characterization of the study sample. The research assistants did not refer problems from the participants in understanding the instructions or questions included in the study. As only 1 participant showed cognitive impairment or psychotic symptoms in the screening, the assessment of these conditions may not be needed in the full trials. This will decrease the time taken for screening interviews, also reducing costs for running the trials.Overall, there was a noticeable trend in all pilot studies for a reduction on depressive symptoms over time, as measured by the PHQ-9. Our approach was categorical, using PHQ-9 cutoff points to decide on caseness and severity. The proposed measure of clinical success, having a PHQ-9 score ≤9 at follow-up (recovery), was considered a stringent but good measure. Nevertheless, all our estimates need to be taken with caution as this pilot study was not powered to detect any statistically significant changes.Other secondary measures of success of the intervention also yielded some results in the expected direction. Disability levels seemed to improve by the end of the intervention. This trend was not as clear for the quality of life measure (EQ-5D).This study also found that participants were satisfied with the intervention and nurse or NA support. Other studies have also shown benefits in the use of collaborative care and task-shifted approaches in the treatment of depression -34. PartTo the best of our knowledge, this is the first study that pilot tested a mobile intervention for symptoms of depression among individuals with chronic conditions in 2 settings of middle-income countries of Latin America. Our samples were small, so the pilot studies were not powered to test the efficacy of the intervention; however, our results were in the predicted direction, and the proportions of treatment success in each sample are useful to calculate full trial sample sizes. We found that many potentially eligible participants could not participate because of difficulties to read or write. This could potentially affect the generalizability of the results. However, with the increasing literacy trend in younger generations in Latin America, these difficulties will decrease, and more people will be able to benefit from similar apps in the future.The technological system, CONEMO, seems feasible to use in these settings with different languages to help patients with diabetes or hypertension and comorbid depressive symptoms. The results from the 3 pilot studies are promising and support the implementation of fully powered trials. The CONEMO intervention will be one of the first evidence-based mobile interventions tested in large samples in 2 different settings in LMICs.


So far the impact on ozone is small, but if these emissions indicate production for foam use much more CFC-11 may be leaked in the future. Assuming such production over 10 years, disappearance of the ozone hole will be delayed by a few years, although there are significant uncertainties. Continued, substantial future CFC-11 emissions of 67 Gg yr−1 would delay Antarctic ozone recovery by well over a decade.The Antarctic ozone hole is decreasing in size but this recovery will be affected by atmospheric variability and any unexpected changes in chlorinated source gas emissions. Here, using model simulations, we show that the ozone hole will largely cease to occur by 2065 given compliance with the Montreal Protocol. If the unusual meteorology of 2002 is repeated, an ozone-hole-free-year could occur as soon as the early 2020s by some metrics. The recently discovered increase in CFC-11 emissions of ~ 13 Gg yr The Antarctic ozone hole is decreasing in size due to policies implemented following the Montreal Protocol. Here, model simulations show that if recently discovered increase in unreported CFC-11 emissions continue, they could delay the recovery of the ozone hole by well over a decade. Following controls on the production of the long-lived halocarbons that transport chlorine and bromine to the stratosphere, atmospheric concentrations of most of them are now decreasing3, and the ozone layer is expected to recover over the course of this century4. Decreases in the stratospheric loading of chlorine and bromine have been observed6, and there are signs of a consequent increase in ozone in the upper stratosphere and the total column8.Depletion of the stratospheric ozone layer by chlorine and bromine species has been a major environmental issue since the early 1970s4. From these metrics, it is clear that the hole has also stopped increasing in size and there are signs of recovery12. However, even with full compliance with the Montreal Protocol, it is expected that the Antarctic ozone hole will persist for many decades into the future.The most significant signal of anthropogenic ozone depletion occurs in the Antarctic in spring—the so-called Antarctic ozone hole. The size of the hole is typically quantified using a range of metrics including minimum column ozone, area contained within a particular value of column ozone and ozone mass deficit13, using results from the chemistry–climate modelling initiative (CCMI) as input to the 2018 WMO Assessment4. They used results from 20 coupled chemistry–climate models (CCMs) to make a best estimate of the dates at which future ozone levels would return to their 1980 values for polar, mid-latitude and tropical regions. Based on this they reported that October column ozone in Antarctic (60oS–90oS) would return to 1980 values in 2060 (with a 1σ uncertainty of 2055–2066) and for March in the Arctic (60oN–90oN) by 2034 (2025–2043). The much earlier return date in the Arctic is due to the smaller depletion and large variability, in conjunction with climate change, which was estimated to have only a small (2-year) effect on the much larger Antarctic loss. This metric of return to a 1980 value is a straightforward concept but it is difficult to estimate and needs to be interpreted with caution. In order to derive values from CCMs, Dhomse et al. (and similar earlier studies14) employed significant smoothing and averaging of the modelled ozone. As a result, the influence of interannual dynamical variability is not included in the uncertainty ranges given above. In addition, in regions where ozone values return asymptotically to the 1980 reference value, a small change in ozone can lead to a large change in return date. Indeed, until and unless ozone values return to the 1980 values then recovery could be deemed not to have happened.The most recent comprehensive assessment of ozone return dates was performed by Dhomse et al.15 recently reported that since the mid-2000s, atmospheric CFC-11 has not been declining as expected, and this discrepancy became particularly striking after 2010, the year production of CFC-11 was reportedly phased out. Their results suggest new emissions16 that linked to unreported production have occurred in recent years. Based on current atmospheric abundances, CFC-11 still contributes about one-quarter15 of anthropogenic chlorine  reaching the stratosphere, so these results could have significant implications for recovery of the ozone layer. Moreover, because nearly all produced CFC-11 eventually escapes to the atmosphere, the impact of this apparent renewed use of CFC-11 on stratospheric ozone ultimately depends on the total amount of new, unreported production, which is currently unknown. If the detected unexpected emissions arise from CFC-11 produced for an emissive use, no large increases in CFC-11 production or banks would be implied, and one would expect the emissions to diminish rapidly if use were terminated. However, because past CFC-11 use was primarily for blowing closed-cell insulating foam that retained most of the CFC17, the detected magnitude of the new, unexpected emissions could imply much larger CFC-11 production quantities and therefore large future emissions.In addition to uncertainties in how to quantify recovery of the ozone layer, a number of factors pose a threat to its expected timescale. Montzka et al.18 reported increasing emissions of dichloromethane (CH2Cl2) based on atmospheric observations from the NOAA global surface network. Using a CCM to investigate different assumptions of the future evolution of CH2Cl2, they estimated a delay in the return of Antarctic ozone to 1980 levels of 5 years for constant future CH2Cl2 concentrations, compared with zero atmospheric CH2Cl2, and up to 30 years for an extreme sensitivity study of constantly increasing CH2Cl2 concentrations. However, those timescales for ozone recovery need to be interpreted with caution as the likelihood for these scenarios to be realised is unknown and the timescales depend on the slow convergence of ozone to a reference recovery baseline. Recently, Fang et al.19 reported increases in the atmospheric abundance of the VSLS chloroform (CHCl3). They also used the model results of Hossaini et al. to estimate the impact of sustained CHCl3 growth on ozone recovery and thereby derived significant delays in 1980 return dates, but these values will have the same caveat of a small change in ozone, causing a large difference in return date, as discussed above. These results need to be reassessed for the impact of realistic amounts of chlorinated VSLS in the context of other changes to chlorine source gases and for a wider range of ozone-hole recovery metrics.Recent studies have also suggested that increasing atmospheric emissions of very short-lived substance species (VSLS), which are not controlled by the Montreal Protocol, may cause a delay to ozone layer recovery. Hossaini et al.Therefore, although ozone recovery is underway, there is uncertainty in how it will progress in the future and how it should be reported. The date for the atmosphere to return to a specified state does not take account of variability in that pathway or on the impact of other transient factors before the final return date. For the Antarctic ozone hole in particular, there are other measures of its size, which may give a different perspective on recovery.In this paper, we use a detailed atmospheric chemical transport model (CTM) to investigate the impact of meteorological variability and non-compliant or uncontrolled chlorine source gas emissions on polar ozone recovery. We quantify the persistence of the Antarctic ozone hole by a range of different metrics, which we show need to be interpreted carefully. We are interested in how long these metrics suggest an ozone hole will occur and how soon, given meteorological variability, we may experience a year without a hole. We also investigate how much longer the Arctic may be susceptible to large ozone loss based on recent extreme meteorology observed in the year 2011 (with extensive occurrence of low temperatures that are conducive to ozone loss). For both cases, we explore the impact on this recovery of the unexpected increase in emissions of CFC-11, under different scenario assumptions. We compare this with the impact of uncontrolled chlorinated VSLS that also enhance polar ozone loss through the increase in stratospheric chlorine. Given the large uncertainties in the CFC-11 emissions related to their source, their main application and future trends, our aim is to test example scenarios and quantify how the impact on ozone varies with the timing and magnitude of the emissions. Our 3D model results can therefore be scaled to assess the impact on column ozone of other total CFC-11 emissions.20 baseline mixing ratio scenario uses a combined atmospheric observation record up to the beginning of 2017 and then future projections. We use this mixing ratio scenario to infer emissions for past years, and obtain good agreement with other estimates15. Considering future mixing ratios, the simple WMO scenario with constant future emissions of CFC-11 of 67 Gg yr−1  produces a much slower decrease in CFC-11 than the baseline scenario, with the global mixing ratio dropping only to 170 ppt in 2080. We have also constructed an additional CFC-11 emission scenario for the recent past based on a constant release fraction from the bank since 200215, which is typically assumed in the creation of future scenarios20 . This scenario indicates the path that would have been expected without these post-2010 emissions from unreported production and also earlier emission changes from 2002, which caused a stabilisation of emissions . We relate this to production by using an estimate of the ratio between rapid initial emission and accumulation in the bank. We then assume a yearly fractional release (leakage) rate for this bank. Finally, we assume a timescale for future rampdown of the unreported production (see Methods). For these 4 parameters, we perform sensitivity tests with the box model to investigate the impact on CFC-11  in mid–late century . In the future, run fODS also uses repeating 2000 meteorology. The net chemical effect of increasing N2O and CH4 causes a gradual decrease in the mean September/October column ozone values, which, in itself, extends the return dates of these metrics compared with the direct effect of halogen decreases22.Different metrics are used to assess the size of the Antarctic ozone hole from observations and model experiments, including September and October mean column ozone from 6000s Fig. . Subsequ23 used a 7-member ensemble of CCM integrations to investigate the range of meteorologically driven annually averaged Antarctic recovery dates. They found the earliest recovery of annual mean ozone to values above those of 1980 at around 2040 and final recovery  in 2060, with an ensemble spread as large as about 15 years. Further analysis of the CCM data gives final recovery dates for October monthly mean column ozone values, averaged from 90oS to 60oS, in the late 2070s, with an ensemble spread ~20 years. This range in return dates is similar to the CTM behaviour for October when run with different meteorologies . This shows that the impact of the additional unexpected emissions to date has likely been very small and highlights the effectiveness of the atmospheric monitoring system for detecting small changes. If these continued emissions are related to production for an immediately emissive use (e.g. a solvent) then the overall impact on ozone may also be small. The potential impact from continued emissions is greater if they have arisen from much larger quantities of production for non-emissive use (e.g. foam). Run R2000_CFC11_B includes additional CFC-11 emissions based on the assumption that post-2010 use of CFC-11 was for non-emissive foam blowing (implying recent production that was around × 6 larger (see Methods) than the unexpected emission increase), and steady elimination of this production over the next 10 years. In this scenario, ozone loss is enhanced, causing a delay in the recovery of ozone of about 2 years no matter which metric is considered (Table −1 (S_CFC11_67) gives a different CFC-11 time dependence and much larger impact compared with the S_CFC11_B scenario. With large, constant emissions, the CFC-11 mixing ratio is maintained at higher levels late in this century  the ozone return dates are brought forward by about 7 years  or short-lived gases (e.g. CH2Cl2) is similar, and depends primarily on the amount of chlorine delivered to the stratosphere. The large mean age-of-air in the polar lower stratosphere (~5 years31) results in a large fractional conversion of most major organic chlorine (and bromine) source gases (whether long or short lived) to inorganic Cly in air transported to this region32.Chlorinated VSLS also affect polar ozone recovery through similar chemical processes. The impact of additional CFC-11 emissions can be compared with the results from run R2000_NoVSLS, which shows that if the stratospheric injection of chlorinated VSLS decreases to zero  and the impact of different CFC-11 and VSLS loadings  data34. The Cly values from the CTM runs  are at the upper end of the CCM range and agree well with MLS. Apart from a few outliers, the total chlorine loading from the CCMs is less variable, indicating that although most CCMs have an overall realistic halogen loading for the past, the partitioning into Cly in the Antarctic vortex is underestimated. In the future, the total chlorine (and Cly) loading in our CTM simulations is larger than the CCMs. This is partly due to the VSLS included in the CTM and partly due to the use of the updated WMO (2018) ODS scenarios  and to have a similar dependence (2000 vs. 2010 meteorology). Based on the different chlorine loadings in the simulations, the delay in ozone return date (Table 18. The simple dependence of ozone return on Cly (whatever its source) is expected because of the role that chlorine plays in polar ozone loss cycles through the ClO + ClO and ClO + BrO catalytic loss cycles36 and the small impact of climate change on Antarctic ozone recovery13. The same dependency of ozone return on Cly is likely to exist for the individual CCMs with realistic polar chemistry shown in Supplementary Figs. Ozone return dates for the total column and at 50 hPa from the individual CCMs tend to correlate with Cl38  will occur well before the mean return date. For a year with disturbed meteorology like that observed in 2002, then, by some metrics, the ozone hole may temporarily recover to 1980 conditions even with the enhanced halogen loadings of the 2020s. Clearly, an early year without an ozone hole (i.e. in the next decade or so) does not indicate full recovery from the effects of ODSs.−1 of a 18-year delay for an extra ~70-ppt CFC-11 are substantial but still do not change the overall trajectory of recovery. Note that for the Antarctic, the expected impact of climate change is only a small (2-year) advance of the 1980 return date13. In comparison, the potential impact of additional CFC-11 emissions can be large.Renewed production and emission of CFC-11 will delay the recovery of the ozone layer. For the Antarctic ozone hole, there is a clear link between the additional amount of chlorine injected into the stratosphere, the additional polar ozone loss and the delay to recovery. However, even if the renewed production so far is for closed-cell foam use, immediate effective measures to stop this could imply a delay of just a few years. Should this renewed production be allowed to continue the impact will be correspondingly more severe, and for the Antarctic ozone hole, can be estimated directly from the chlorine-loading enhancement. The estimates derived here for the large WMO (2018) emission scenario of constant 67 Gg yry loading can be used to apply our results to perturbations of other source gases. In the aged air of the polar lower stratosphere, most major source gases are nearly completely converted to inorganic forms. Therefore, if CFC-12 is being co-produced along with the additional CFC-11 and is largely being contained in a bank, a process that we have not explicitly considered, the impact on Antarctic ozone recovery will scale with chlorine loading supplied by this additional CFC-12 as it is eventually emitted. Minimum co-production rates of CFC-12 in the most common industrial process for producing CFC-11 are ~30%, so this additional chlorine might be substantial39. Similarly, the polar impact of any additional emissions of CCl4, which is not decreasing as rapidly as expected in the atmosphere42, will depend on the resulting increase in stratospheric chlorine.The relationship between Antarctic ozone recovery, emissions and polar Cl18 and our simulation R2000_NoVSLS demonstrate the potential for VSLS to influence timescales for ozone recovery through changing stratospheric chlorine loading. However, the degree to which such potential is realised and the overall significance of VSLS with respect to ozone will depend on both the magnitude and trend of their future emissions. For CH2Cl2, the most abundant chlorinated VSLS, global emissions increased by a factor of ~2 between 2000 (~500 Gg yr−1) and 2016 (~1000 Gg yr−1), as derived from NOAA data4. While such growth may have acted to offset the rate of upper stratospheric HCl decline, by ~15% since the mid-2000s30, firm conclusions on any future impacts require more accurate estimates of likely future emission changes. For VSLS with both mixed emissive and non-emissive applications, notably CH2Cl2, such scenarios could be developed using production information and market analyses.Emissions of VSLS are also a source of stratospheric chlorine. Previous sensitivity studies2, but, with our detailed knowledge of the processes involved, we can now use different metrics to measure recovery (e.g. ref. 11). In this paper, we have shown that three decades since it was ratified, its continued success does face some challenges from recent unreported CFC-11 production. However, with swift action to curb this production and any other, the long-term success of the protocol will be ensured.The Montreal Protocol is rightly seen as a seminal international agreement that has successfully led to decreasing levels of atmospheric chlorine and bromine and early signs of ozone recovery. For the Antarctic ozone hole, this recovery is best measured by metrics related to the extent of ozone loss in September rather than October and by the decrease in depletion by a certain date, rather than return to the 1980 value. Previous historic use of October metrics was related to datasets used to discover the ozone hole43. The model has been widely used in previous studies45 and simulates stratospheric ozone well. The model includes a detailed treatment of stratospheric chemistry, including a full description of processes related to polar ozone depletion. The model is forced by ERA-Interim reanalyses provided by the European Centre for Medium-Range Weather Forecasts (ECMWF)46. The model was integrated in series of experiments at a horizontal resolution of 2.8o × 2.8o with 32 levels from the surface to ~60 km. The runs were forced by observed and predicted surface mixing ratios of long-lived source gases from 1955 to 2100. The upper troposphere mixing ratios of short-lived chlorine species were specified using estimates from 2000 to 201630, with constant values before and after this period.We have used the TOMCAT/SLIMCAT offline three-dimensional (3D) CTM to calculate the impact of chlorine scenarios and meteorological variability on stratospheric ozone4. The surface CH4 and N2O scenarios are taken from the Special Report on Emissions (SRES) scenario A1b . This approach ignores the impact of climate change on stratospheric temperatures and circulation, which will become increasingly important as the simulations progress. Climate change certainly has an important impact on ozone recovery in the upper stratosphere where cooling acts to increase ozone and adds to the effect of decreasing chlorine4. However, in this paper, we focus on polar lower stratospheric ozone loss where the impact of temperature trends is less important13. Moreover, by using this approach, we ensure that the model results have realistic polar meteorological conditions that are important for accurate simulation of ozone loss. By running the model with different repeating analysis years we aim to span the effects of climate change on polar vortex dynamics.We drive the future model simulations with repeating meteorological analyses from previous years but with time-dependent ODS concentrations, which for the control run are taken from the baseline A1 scenario of WMO (2018)−1 . This is used as a sensitivity run to examine how the co-emission of longer-lived CFC-12 (lifetime 102 years47) will affect the model projections. Run R2000_NoVSLS was the same as R2000 but with post-2018 emissions of short-lived chlorine species set to zero (corresponding to 114 ppt less chlorine in the stratosphere). Finally, we performed a simulation fODS that was identical to R2000 but with halogenated ODS values constant at 1960 values. The length of the sensitivity runs varied depending on the rate of recovery to 1980 ozone values.Model simulation R2000 was integrated from 1955 to 2080 using repeating 2000 meteorology and time-dependent ODS concentrations  data. These data combine ozone total column retrievals from various UV–nadir, limb and occultation satellite sensors. A key feature of these data is that climate data records (CDR) and interim-CDR parts of each product are generated using the same software and algorithms. Total ozone data used here are so-called level 4 data that combine data from 15 satellite instruments and fill the missing values using data assimilation system. These data are available from January 1970 to present at 1.0o × 1.0o resolution and are obtained from https://cds.climate.copernicus.eu/cdsapp#!/dataset/satellite-ozone?tab = overviewTo compare with our past model simulations we use various satellite data products. For total column comparisons, we use the NASA Solar Backscatter Ultra-Violet SBUV (Version 8.6) merged dataset that is constructed by merging individual SBUV/SBUV/2  satellite datasets4 to an estimate of annual emissions, assuming that the surface values correspond to a global mean. This gave good agreement with independent estimates from a multi-box model15. Our estimated emissions were then used as a basis for estimating emissions from non-reported production of CFC-11 since 2010 and for future example sensitivity scenarios, bearing in mind the large uncertainties related to the new CFC-11 emissions and how they will vary. We use 13 ± 5 Gg yr−1 as the estimate of emissions related to unreported production in recent years15, which we relate to an assumed total production and project forward in time, assuming that the CFC-11 is produced for use in closed-cell foams. Scenario S_CFC11_B assumes that 15% of CFC-11 produced is released immediately49 (ratio 5.66:1), followed by 3.5% yr−1 (ref. 15) as leakage from the bank. We assume that through policy action the unreported production will rampdown to zero over 10 years. Further box model runs were performed to examine the sensitivity to these assumptions , which seems inconsistent with likely applications. Therefore, we adopt the representative range of ×3 to ×9. The fractional release was varied from 2.5 to 4.5% yr−1, which spans the upper range given in ref. 49. Finally we varied the time for the production rampdown from 5 to 20 years. A further future scenario was defined by taking the WMO (2018) scenario with an additional 67 Gg yr−1 of emissions . This is likely unrealistic but it serves as a 3D model sensitivity simulation. Scenario S_CFC11_B leads to around 45-pptv additional chlorine in the atmosphere over the next few decades (Supplementary Fig. We have related emissions of CFC-11 to mean atmospheric mixing ratios using a global 1-box model Figs. . The mod ratio 5.6:1, follSupplementary InformationPeer Review File


Saccharomyces cerevisiae population exhibiting two phenotypic expression states of the PDR5 gene. This gene grants multidrug resistance by transcribing a membrane transporter capable of pumping out cytotoxic compounds from the cell. Utilizing fluorescent markers, single cells were isolated and trapped, then their growth rates were measured in two on-chip environments: rich media and media dosed with the antibiotic cycloheximide. Approximating growth rates to first-order, we assessed the fitness of individual cells and found that those with low PDR5 expression had higher fitness in rich media whereas cells with high PDR5 expression had higher fitness in the presence of the drug. Moreover, the drug dramatically reduced the fitness of cells with low PDR5 expression but had comparatively minimal impact on the fitness of cells with high PDR5 expression. Our experiments show the utility of this imaging-integrated microfluidic cell volume sensor for high-resolution, single-cell analysis, as well as its potential application for studies that characterize and compare the fitness and morphology of individual cells from heterogeneous populations under different growth conditions.An imaging-integrated microfluidic cell volume sensor was used to evaluate the volumetric growth rate of single cells from a  For the cell, phenotypic output can reveal much about the constituents that govern transcription, translation, and signaling. One such phenotypic output is cellular growth rate. Growth rates have been shown to be directly related to gene expression, metabolism, stress response, and the cell cycle3. Indeed, precise coordination between growth and the cell cycle is necessary for proper cell division4, and its disruption can even lead to carcinogenesis5. Yet, continued research efforts are still needed to attain a complete and comprehensive understanding of cell cycle regulation7. We contend that the development of more precise measurement platforms capable of monitoring cellular growth is crucial in further expanding this research area. Conventional methods often utilize bulk population-level measurements because they are relatively easy and well-established. However, the measurement of population-level averages masks subtleties at the single-cell level9 where heterogeneity can arise even for clonal populations growing in a homogeneous environment11. For example, it was only through the measurement single-cell growth rates that Son et al.12 were able to determine that the cell cycle in some mammalian cells is regulated by a critical growth rate as opposed to a critical cell size. But even when cell cycle is regulated by size, such as in yeast, it can still be coupled to the cellular growth rate13. In addition, if mathematical descriptions of cellular function are to be formulated, individual cell data will be needed for precise validation of systems biology models14. Chip-based biosensors are uniquely poised to tackle these issues and have seen rising use in single-cell characterization16, including label-free and non-invasive methods like impedance cytometry18 and suspended microchannel resonator (SMR) mass sensors19.Cell biology has traditionally employed the reductionist approach, focusing on the function of individual genes and proteins. But it has become increasingly necessary to understand how cells behave on a systems level as more than the sum of its parts20, osmotic stress21, and nutrient availability22. For example, one study has shown that loss of growth control under low nutrient availability can lead to tumorigenesis23. In situ variation of the growth environment in particular enables experimental evolution at the fundamental cell level and facilitates the study of phenotypic plasticity24, for which growth rate can serve as a marker25. This of course includes the characterization of drug effects, where individual cell growth and cell cycle data can support models of growth dynamics in response to drug treatment26. This is especially important as pathogenic microbes are increasingly evolving resistance to antibiotics27 and cancer cells are known to possess mechanisms of multidrug resistance (MDR)28. Another environment-dependent evolutionary strategy that can be studied is bet-hedging, where isogenic populations cope with environmental fluctuations by stochastically producing phenotypic heterogeneity with different fitness advantages29. A key aspect of bet-hedging is that because the individual cells randomly switch between phenotypes, they inherently generate heterogeneous populations, which can render bulk fitness measurements misleading, as opposed to single-cell measurements.A crucial factor in understanding cellular function is the strong coupling to environmental conditions, such as salinitySaccharomyces cerevisiae) exhibiting two phenotypic expression states of the PDR5 gene, high-expressing (HE) and low-expressing (LE). This gene encodes a transmembrane pump that is part of a MDR network30 and is capable of actively removing compounds from the cell31. It is one of the best-studied ATP-binding cassette transporters32 and is a homologue of the human gene MDR1 implicated in many drug resistant cancers33. Because PDR5 grants multidrug resistance, the maintenance of a bimodal population could be accompanied by fitness costs.This paper highlights an imaging-integrated microfluidic cell volume sensor that is able to track growth at the single cell level while controlling the surrounding micro-environment. In this study, we investigate a strain of budding yeast  fabricated using standard photolithography and multilayered soft lithography34. The PDMS body is oxygen plasma-bonded to a piece of glass on which the gold planar electrodes were patterned. Integrated valves allow the deformation of the adjacent fluidic microchannels. As shown in Fig. 36. Metal deposition was done using electron beam evaporation and the electrode geometry was patterned using photolithography. Excess metal was then etched using aqua regia and hydrofluoric acid. Each electrode is 20 μm wide, 50 nm thick with a 5 nm titanium adhesion layer, and their inner edges are 30 μm apart. The sensing channel spanned by the electrodes is 25 μm wide and 20 μm tall when the valve V3 is not compressed.The microfluidic cell volume sensor used to quantify volumetric growth in this study was adapted from a similar platform validated previously38. The input signal passes through a unity gain low-noise preamplifier before going to the chip. The output signal is converted to a voltage using a transimpedance amplifier and then fed to a lock-in amplifier. When completely sealing the sensing channel, valves V1 and V2 .During experiments, the chip is maintained at 30 °C and mounted on the microscope stage. Bright-field imaging was done in transmission mode where the transparency of PDMS allowed for clear illumination. The sensing electrodes are driven with an AC signal of amplitude 300 mV and frequency 100 kHz. The frequency was chosen so that the effects of the electrode polarization impedance were minimized  were added to YPgal; this represents the rich media environment in this study. The 50 mM concentration of NaCl provides an optimized ionic current for measurements whilst being low enough to not affect growth. Cycloheximide was added to YPgal with 50 mM NaCl and 0.5% BSA to form the drug environment in this study, with a final cycloheximide concentration of 0.1 µg/mL.The base culture media used was yeast extract-peptone-galactose , which consists of 2% w/v yeast extract, 4% w/v bacteriological peptone, and 2% w/v galactose, dissolved in DI-H600 of 0.1 to 0.4. For the high-expressing cells, plates were streaked using strictly log-phase cultures (and OD600 not exceeding 0.4). Samples of the high-expressing cells used for the on-chip experiments were then taken from log-phase cultures inoculated using only the smallest, slowest-growing colonies, as these colonies were formed from an initial high-expressing cell. All the single cells were randomly chosen from the liquid culture and so were not cell cycle synchronized.For the low-expressing cells used, initial colonies were prepared by streaking agar plates using cells from a frozen stock that were cultured in YPgal and incubated for up to 24 hours at 30 °C. The low-expressing cell culture was inoculated from one of these colonies, incubated overnight in YPgal, and then diluted once. The sample was taken during log-phase, once the diluted culture reached an ODPDR5 gene, along with an 8 amino acid linker to maximize fluorescence. A KanMX cassette was also placed downstream of yEGFP for selection of successful recombinants. The parent BY4741 strain  was used as a background for the recombination. An area of homology to the C-terminal sequence of PDR5 was amplified using primers that would exclude the stop codon. Another area of homology upstream of the stop codon was also amplified to ensure efficient integration of the construct in the correct position. The two homologous sequences, as well as the yEGFP coding sequence and the KanMX cassette, were fused to each other in a pairwise fashion using overlap extension PCR. The construct was then transformed into the BY4741 strain.Yeast enhanced green fluorescent protein (yEGFP) was fused to Pdr5 by using homology-based transformation assisted recombination (TAR) cloning to insert a yEGFP coding sequence directly downstream of the Yeast cultures in log-phase growth were diluted 10X in 50 mM sodium citrate buffer. Flow cytometry was performed with a CyAn ADP 9 Analyzer  using a 488 nm solid-state laser to excite the yEGFP fluorophore. Fluorescence emission was detected with a 530/40 filter. All Flow Cytometry Standard file formats (.fcs) were analyzed using MATLAB  with a custom FCS extraction and analysis script.PDR5 expression, is higher than the auto-fluorescence of wild type cells. Using flow cytometry to characterize the PDR5 expression, we found that for log-phase cells grown in YPgal, the majority (approximately 94%) exists in a low-expressing (LE) state, while a small subpopulation (approximately 6%) persists in a high-expressing (HE) state with a PDR5 expression level that is about 10 times higher. This difference in PDR5 expression is also evident in the fluorescence intensity of the HE cells compared to the surrounding LE cells shown in Fig. PDR5 expression in budding yeast.Tagging Pdr5 with yEGFP successfully allowed us to observe its expression pattern. Figure In order to evaluate the fitness of each phenotype and their corresponding disparities, the aforementioned microfluidic cell volume sensor was used. Cells were flowed into the microfluidic device in the rich media they were cultured in, a target was then trapped in the middle sensing channel and the sieve valves were closed to seal it off from the two bypass flow channels. A fluorescence image was then captured to ascertain the expression level  so that growth rates could be evaluated. Other studies with budding yeast have used concentrations up to three orders of magnitude higher40, how a necrotic death is prompted only after prolonged exposure is unclear. As for why there is an abrupt drop in the measured volume, we note that at the relatively low driving frequency of 100 kHz, an intact cell membrane contributes a large capacitive impedance44, but a sudden loss of membrane integrity would result in an increase of ionic current through the volume occupied by the cell and a corresponding decrease in the magnitude of the current pulse. This is supported by the fact that necrotic death is characterized by a rapid increase in the permeability of the plasma membrane45. Thus, this platform allows for rapid detection of changes to the dielectric properties of the cell membrane as well as the characterization of subtle changes to membrane integrity over time, which can be useful for studying drugs that target membrane permeability. However, the dielectric properties of cell death are better characterized by impedance spectroscopy46. We also observed that the mean growth rate following the shrinking event is greater than the mean single-cell growth rate of the LE cells in drug while being less than their mean collective growth rate before the shrinking occurred. For this, it is understandable that cell contents would leak into the environment given the loss of membrane integrity, but considering the comparison that was just given, it may be possible that the mother was also reabsorbing some of the matter in the bud.The triggers and mechanisms underlying the shrinking event observed for LE cells in drug remain unclear. Cycloheximide is a broad inhibiter of protein synthesis in eukaryotes, thus it should interfere with all phases of the cell cycle. However, some of the LE cells were able to begin division shortly after introduction of the cycloheximide-dosed media. This implies that despite a toxic presence, they successfully proceeded into S phase. This next phase is not immediately aborted and is maintained for up to several hours until an abrupt shrinking event occurs over a period of seconds. Though the effects of cycloheximide could be playing a role in impeding and delaying apoptosis42. The G1 checkpoint can be effectively obstructed by blocking the translation of important proteins for the transition into S phase48. But in three of the cases where division had been initiated at the beginning of the experiment, a second bud was observed emerging later on despite the previous death of the first daughter. However, their viabilities were undetermined. Since the presence of cycloheximide induces increased PDR5 transcription49, it was possible that those mother cells were able to produce more efflux pumps over time so as to expel enough cycloheximide and manage a degree of protein synthesis that allowed for procession through the G1 checkpoint. However, none of this culminated in confirmed reproductive success over the observation period.For the LE cells that did not initiate division at the beginning, they were never observed to do so throughout the entirety of the experiment. It is most likely that the insufficient number of efflux pumps resulted in a concentration of cycloheximide within the cell that was able to keep it arrested in the G1 phasePDR5 expression cells, the entire population avoids the risk of complete extinction in the event of a sudden shift to a highly toxic environment where greater resistance is needed. However, it is not entirely clear if the over-expression of PDR5 is the sole cause of the slower growth, though it could be a contributing factor as excessive protein production taxes cellular resources. In addition, as PDR5 encodes a transmembrane efflux pump, it may also lead to indiscriminate removal of nutrients or other necessary compounds from within the cytosol. This pump also requires ATP hydrolysis to function, thus an over-abundant number of them would become an onerous energy expenditure for the cell. In fact, a recent study found that in S. cerevisiae, excessive expression of proteins containing membrane-protruding regions resulted in a very high fitness cost50. But this coupling of high PDR5 expression to slower growth is perhaps to be expected as it is consistent with studies that have shown slowed growth is correlated to stress resistance and response52.The evidence presented here demonstrates that each of the two phenotypes confers a different reproductive fitness advantage depending on the environment. Thus, by maintaining a small subpopulation of high 29, and the mechanisms which control and give rise to the observed phenotypic heterogeneity in PDR5 expression still remain unclear. While the yeast used is of a single strain, it would be necessary to determine that the high PDR5 expression is not due to unexpected chromosomal and/or mitochondrial mutations. Given that stochastic processes underlie bet-hedging53, random switching between the phenotypic states would also need to be observed. Nonetheless, the fitness requirement of bet-hedging was successfully quantified using this platform, and is therefore fully applicable to such studies. This method also offers the advantage of being able to check the phenotypic state of single cells at any stage via fluorescence, and since bet-hedging individuals inherently switch states, bulk measurements could be misleading about the true nature of such phenotypes.It is worth noting that although the observed fitness disparity is required by the evolutionary strategy of bet-hedging, there are also additional criteria that need to be metPDR5 gene exhibited by a S. cerevisiae population have associated environment-dependent fitness disparities. Through control of the on-chip environment, we found that while the low-expressing cells had higher fitness in rich media, severe deficiencies were apparent in the presence of cycloheximide. The high-expressing cells, however, were able to cope equally well in both conditions, and had higher relative fitness in the drug environment. While we measured fitness as a phenotypic output of gene expression, these results underscore the importance of growth rate as a biophysical marker for cells as well as its potential to serve as a parameter for probing the systems level effects of engineered perturbations. The interplay between cell growth, the cell cycle, and environmental conditions is complex and interconnected, and we hope the applicability of our device for studying these relationships from the single-cell perspective will help to unravel these complexities.Using an imaging-integrated microfluidic cell volume sensor capable of measuring single-cell growth rates, we showed that two phenotypic expression states of the Supplementary Information