Iterative approach to computational enzyme design

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes

Application of Photovoice with Focus Groups to Explore Dietary Behaviors of Older Filipino Adults with Cardiovascular Disease

Filipino Americans have high rates of cardiovascular diseases (CVD). This study explored the dietary behaviors, a modifiable risk factor, of Filipinos with CVD. Filipinos with CVD were recruited and trained to do Photovoice. Participants took photos to depict their “food experience,” defined as their daily dietary activities. Participants then shared their photos during focus groups. Focus group transcripts were analyzed using an iterative, grounded theory approach. Among 38 Filipino participants, the mean age was 70 years old and all were foreign-born. Major themes included efforts to retain connection to Filipino culture through food, and dietary habits shaped by cultural health beliefs. Many believed that traditional dietary practices increased CVD risk. Receiving a CVD diagnosis and clinician advice changed their dietary behaviors. Household members, the physical environment, and economic constraints also influenced dietary behaviors. Photovoice is feasible among older Filipinos and may enhance understanding of drivers of dietary behaviors

A PP2A-B55-Mediated Crosstalk between TORC1 and TORC2 Regulates the Differentiation Response in Fission Yeast

Extracellular cues regulate cell fate, and this is mainly achieved through the engagement of specific transcriptional programs. The TORC1 and TORC2 complexes mediate the integration of nutritional cues to cellular behavior, but their interplay is poorly understood. Here, we use fission yeast to investigate how phosphatase activity participates in this interplay during the switch from proliferation to sexual differentiation. We find that loss of PP2A-B55$^{Pab1}$ enhances the expression of differentiation-specific genes and leads to premature conjugation. $\textit{pab1}$ deletion brings about a transcriptional profile similar to TORC1 inactivation, and deletion of $\textit{pab1}$ overcomes the repression of differentiation genes in cells overexpressing TORC1. Importantly, we show that this effect is mediated by an increased TORC2-AKT (Gad8) signaling. Under nutrient-rich conditions, PP2A-B55$^{Pab1}$ dephosphorylates Gad8 Ser546, repressing its activity. Conversely, TORC1 inactivation upon starvation leads to the inactivation of PP2A-B55$^{Pab1}$ through the Greatwall-Endosulfin pathway. This results in the activation of Gad8 and the commitment to differentiation. Thus, PP2A-B55$^{Pab1}$ enables a crosstalk between the two TOR complexes that controls cell-fate decisions in response to nutrient availability.We thank Dominique Helmlinger and Sergio Moreno for sharing unpublished results and strains, and for stimulating discussion. We thank Janni Petersen, Ronit Weisman, Kazuhiro Shiozaki, Mitsuhiro Yanagida, and Hisao Masukata for strains, constructs, and antibodies. We thank Toni Hurtado and Beata Grallert for critical reading of the manuscript. This work was supported by NFR FRIMEDBIO grant 214049. M.P. is the recipient of a Kreftforeningen postdoctoral fellowship (grant 5843744). N.C. has received funding from the European Union Seventh Framework Programme (FP7-PEOPLE-2013-COFUND) under grant agreement 609020 - Scientia Fellows. J.M. was supported by BBSRC research grants BB/N007697/1 and BB/M021483/1

Chronic breathlessness and sleep problems: a population-based survey

ObjectivesThis study aimed to explore the relationship (presence and severity) between chronic breathlessness and sleep problems, independently of diagnoses and health service contact by surveying a large, representative sample of the general population.SettingAnalysis of the 2017 South Australian Health Omnibus Survey, an annual, cross-sectional, face-to-face, multistage, clustered area systematic sampling survey carried out in Spring 2017.Chronic breathlessness was self-reported using the ordinal modified Medical Research Council (mMRC; scores 0 (none) to 4 (housebound)) where breathlessness has been present for more than 3 of the previous 6 months. 'Sleep problems-ever' and 'sleep problem-current' were assessed dichotomously. Regression models were adjusted for age; sex and body mass index (BMI).Results2900 responses were available (mean age 48.2 years (SD=18.6); 51% were female; mean BMI 27. 1 (SD=5.9)). Prevalence was: 2.7% (n=78) sleep problems-past; 6.8% (n=198) sleep problems-current and breathlessness (mMRC 1-4) was 8.8% (n=254). Respondents with sleep problemspast were more likely to be breathless, older with a higher BMI and sleep problems-present also included a higher likelihood of being female.After adjusting for age, sex and BMI, respondents with chronic breathlessness had 1.9 (95% CI=1.0 to 3.5) times the odds of sleep problems-past and sleep problems-current (adjusted OR=2.3; 95% CI=1.6 to 3.3).ConclusionsThere is a strong association between the two prevalent conditions. Future work will seek to understand if there is a causal relationship using validated sleep assessment tools and whether better managing one condition improves the other

Impact of long‐term management with sleep medications on blood pressure: An Australian national study

OnlinePublBackground: There is mixed evidence about the impact of long-term management with hypnotic medications on blood pressure (BP). Aim: To estimate the effect of short- and long-term management with benzodiazepine and z-drugs (BZD) on BP. Method: Open cohort study using deidentified electronic health records of 523,486 adult regular patients (42.3% males; mean age 59.0 ± 17.0 years) annually attending 402 Australian general practices between 2016 to 2018 (MedicineInsight database). Average treatment effects (ATE) of recorded incident BZD prescriptions in 2017 on systolic (SBP) and diastolic (DBP) BP after starting these prescriptions were computed using augmented inverse probability weighting (AIPW). Results: In 2017, 16,623 new cases of short-term management with BZD and 2532 cases of long-term management with BZD were identified (incidence 3.2% and 0.5%, respectively). The mean BP among those not treated with BZD (reference group) was 130.9/77.3 mmHg. Patients prescribed short-term BZD showed a slightly higher SBP (ATE 0.4; 95% CI 0.1, 0.7) and DBP (ATE 0.5; 95% CI 0.3, 0.7), while those on long-term BZD prescriptions showed lower SBP (ATE -1.1; 95% CI −2.0, −0.2), but no effect on DBP (ATE −0.1; 95% CI −0.8, 0.5). However, long-term BZD prescriptions showed a stronger BP-lowering effect among patients aged 65+ years (SBP ATE −2.5 [95% CI −3.8, −1.3]; DBP ATE −1.0 [95% CI −1.7, −0.2]), but almost no effect was observed among younger patients. Conclusion: Long-term management with BZD had a BP-lowering effect among older patients. These findings add new evidence to current recommendations on limiting long-term BZD management in the elderly.Mumtaz Begum, David Gonzalez-Chica, Carla Bernardo, Nigel Stock

Epidemiology of arthritis, chronic back pain, gout, osteoporosis, spondyloarthropathies and rheumatoid arthritis among 1.5 million patients in Australian general practice: NPS MedicineWise MedicineInsight dataset

Background: Previous estimates for the prevalence of musculoskeletal conditions (MSK) and chronic pain in Australia have been based on self-report. We aimed to determine the prevalence and distribution of arthritis, chronic back pain, gout, osteoporosis, spondyloarthropathies and rheumatoid arthritis and current consultations for chronic pain among adults attending Australian general practice, and describe their distribution according to sociodemographic characteristics and presence of co-morbidities. Methods: We investigated 1,501,267 active adult patients (57.6% females; 22.5% ≥65y) evaluated between 2013 and 2016 and included in the MedicineInsight database (a National Prescribing Service MedicineWise program), a large general practice data program that extracts longitudinal de-identified electronic medical record data from ‘active’ patients in over 550 practices. Three main groups of outcomes were investigated: 1) “prevalence” of arthritis, chronic back pain, gout, osteoporosis, spondyloarthropathies, and/or rheumatoid arthritis between 2000 and 2016; 2) “current” diagnosis/encounter for the same conditions occurring between 2013 and 2016, and; 3) “current” consultations for chronic pain of any type occurring between 2013 and 2016. Results: The combined “prevalence” of the investigated MSK (diagnosis between 2000 and 2016) among adults attending Australian general practice was 16.8% (95%CI 15.9;17.7) with 21.3% (95%CI 20.2;22.4) of the sample consulting for chronic pain between 2013 and 2016. The investigated MSK with the highest “prevalence” were arthritis (9.5%) and chronic back pain (6.7%). Patients with some of these MSK attended general practices more frequently than those without these conditions (median 2.0 and 1.0 contacts/year, respectively). The “prevalence” of the investigated MSK and “current” consultations for chronic pain increased with age, especially in women, but chronic pain remained stable at 22% for males aged > 40 years. The investigated MSK and chronic pain were more frequent among those in lower socioeconomic groups, veterans, Aboriginal and Torrent Strait Islanders, current and ex-smokers, and patients with chronic obstructive pulmonary disease or heart failure. Conclusions: The investigated MSK are more frequent among lower socioeconomic groups and the elderly. Based on information collected from adults attending Australian general practices, MedicineInsight provided similar estimates to those obtained from population-based studies, with the advantage of being based on medical diagnosis and including a national sample.David Alejandro González-Chica, Simon Vanlint, Elizabeth Hoon and Nigel Stock

Impact of funding influenza vaccination on coverage among Australian children: a national study using MedicineInsight, a large general practice database

Published Online 7 October 2019Influenza contributes to morbidity and mortality worldwide. Children are at a higher risk of influenza-related complications and vaccination promotes direct protection and limits transmission. This study aimed to explore influenza vaccination coverage among children in Australian general practice from 2015 to 2018, and patterns in coverage before and after the implementation of state-funded immunization programs. Data from 196,520 ‘active’ patients (3+ consultations in two consecutive years) aged <5 years from 542 Australian general practices were included (MedicineInsight database). Logistic regression models were used to identify associations between vaccination with patient and practice characteristics. The overall vaccination coverage increased more than five times from 2015 (3.9%) to 2018 (19.6%) and varied among states. Children attending practices located in the wealthiest areas were more likely to receive the vaccine and appeared to benefit most from the funding, as the increase in coverage from 2017 to 2018 was greater among them than those attending practices in the least advantaged areas (17 vs. 11 percentage points, respectively). This relationship was not evident when analyzing the patient’s socioeconomic level. In conclusion, free influenza vaccinations increase coverage in at-risk populations. Promotional campaigns may be required to maintain higher coverage and target practices located in low-income areas.Carla De Oliveira Bernardo, David Alejandro González-Chica, and Nigel Stock

Burden and health-related quality of life of eating disorders, including Avoidant/Restrictive Food Intake Disorder (ARFID), in the Australian population

Background: Little is known about the epidemiology and health related quality of life (HRQoL) of the new DSM-5 diagnoses, Binge Eating Disorder (BED) and Avoidant/Restrictive Food Intake Disorder (ARFID) in the Australian population. We aimed to investigate the prevalance and burden of these disorders. Methods: We conducted two sequential population-based surveys including individuals aged over 15 years who were interviewed in 2014 (n = 2732) and 2015 (n =3005). Demographic information and diagnostic features of DSM-5 eating disorders were asked including the occurrence of regular (at least weekly over the past 3 months) objective binge eating with levels of distress, extreme dietary restriction/fasting for weight/shape control, purging behaviors, overvaluation of shape and/or weight, and the presence of an avoidant/restrictive food intake without overvaluation of shape and/or weight. In 2014 functional impact or role performance was measured with the 'days out of role' question and in 2015, Health Related Quality of Life (HRQoL) was assessed with the Short Form -12 item questionnaire (SF-12v1). Results: The 2014 and 2015 3-month prevalence of eating disorders were: anorexia nervosa-broad 0.4% (95% CI 0.2-0.7) and 0.5% (0.3-0.9); bulimia nervosa 1.1% (0.7-1.5) and 1.2% (0.9-1.7); ARFID 0.3% (0.1-0.5) and 0.3% (0.2-0.6). The 2015 3-month prevalence rates were: BED-broad 1.5% (1.1-2.0); Other Specified Feeding or Eating Disorder (OSFED) 3.2 (2.6-3.9); and Unspecified Feeding or Eating Disorder (UFED) 10.4% (0.9-11.5). Most people with OSFED had atypical anorexia nervosa and majority with UFED were characterised by having recurrent binge eating without marked distress. Eating disorders were represented throughout sociodemographic groups and those with bulimia nervosa and BED-broad had mean weight (BMI, kg/m²) in the obese range. Mental HRQoL was poor in all eating disorder groups but particularly poor for those with BED-broad and ARFID. Individuals with bulimia nervosa, BED-broad and OSFED-Purging Disorder also had poor physical HRQoL. ARFID and bulimia nervosa groups had lower role performance than those without an eating disorder. Conclusions: Whilst full spectrum eating disorders, including ARFID, were less common than OSFED or UFED, they were associated with poor mental HRQoL and significant functional impairment. The present study supports the movement of eating disorders in to broader socio demographic groups including men, socio-economic disadvantaged groups and those with obesity.Phillipa Hay, Deborah Mitchison, Abraham Ernesto Lopez Collado, David Alejandro González-Chica, Nigel Stocks and Stephen Touy

Pneumococcal vaccination uptake among patients aged 65 years or over in Australian general practice

In Australia, pneumococcal vaccine is provided free to all adults aged ≥65 years and Indigenous people aged 15-65 years, and is subsidised for non-Indigenous adults <65 years of age with risk factors. This study aimed to explore pneumococcal vaccination uptake in older patients attending 550 Australian general practice from 2010-2017 by patient sociodemographics, presence of comorbidities and practice characteristics. Study 1: a cross-sectional analysis of 'active' patients aged ≥65 years in each year was performed to calculate annual pneumococcal vaccination uptake. Study 2: a cohort of 58,589 'every year' patients aged 60-65 years in 2010 was analysed to identify the number of patients immunised during the study period. Logistic regression models assessed associations between vaccination, patient and practice characteristics. Annual pneumococcal vaccine uptake varied by patient's age (65-74 or ≥75 years), presence of comorbidities and regularity of practice visits (range 36% to 76%), and it declined slowly from 2011-2016 amongst all groups. Cohort analyses showed that 69% of those aged 60-65 years in 2010 had a recorded pneumococcal vaccination by 2017 (peak age of vaccination = 66 years), and vaccination was more likely among those with comorbidities, ex-smokers and frequent attenders to practices. Findings demonstrate that the NPS MedicineInsight database provides estimates of vaccination uptake consistent with past surveys, reproducible every year and at low cost. It has the advantage of additional clinical information compared to the Australian Immunisation Register. Whilst vaccination uptake was adequate among 'every year' patients, interventions are needed to improve pneumococcal vaccination for all older Australians.Oliver Frank, Carla De Oliveira Bernardo, David Alejandro González-Chica, Kristine Macartney, Robert Menzies and Nigel Stock