Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.

Elexacaftor-Tezacaftor-Ivacaftor in 2 cystic fibrosis adults homozygous for M1101K with end-stage lung disease.

Elexacaftor-tezacaftor-ivacaftor (ETI) therapy is shown to improve the health of individuals with cystic fibrosis (CF) who have the F508del variant. There are in vitro studies showing benefit with ETI for select rare CF variants. Limited data exists on the use of ETI in individuals with rare CF variants, particularly in those with advanced lung disease. We present 2 cases of CF individuals homozygous for the rare M1101K variant with end-stage lung disease who demonstrated sustained improvements in lung function, pulmonary exacerbation frequency, respiratory symptoms, and body mass index after 6 months of ETI treatment - similar to that expected with F508del.

Microbiology sampling in non-cystic fibrosis bronchiectasis cases from northern Alberta.

Non-cystic fibrosis bronchiectasis (NCFB) is a chronic respiratory disease resulting in chronic cough, thick sputum, and lower airway microbial colonization, akin to patients with cystic fibrosis (CF). NCFB is a common, yet under recognized entity which inflicts significant morbidity and mortality particularly to older individuals, with a rising prevalence in the developed world. Given that sputum cultures are a non-invasive method to characterize the lower airway microbiota in NCFB patients, for which pathogenic organisms are associated with worsened outcomes, we sought to characterize the microbiological pattern and clinical outcomes associated with sputum culture in a cohort of NCFB patients from Western Canada. A total of 20 subjects were prospectively recruited from various bronchiectasis clinics across the Greater Edmonton area. A retrospective chart review and a symptoms questionnaire was performed, gathering information not limited to symptoms, comorbidities, exacerbations, hospitalizations, sputum production, and sputum culture results over the prior 5 years. Subjects reported frequent hospitalization alongside a significant burden of symptoms. A large majority of sputum cultures grew pathogenic organisms such as Haemophilus influenzae and Pseudomonas aeruginosa. We also note the considerable waste and inefficiency associated with sputum cultures, outlining areas for which this important diagnostic modality can be improved. Accurate characterization of the airway microbiota alongside efficient delivery of health services are key to ensuring the proper treatment of individuals with NCFB, given their high disease burden and frequent hospitalization.

Career and life development intervention for non-engaged youth: Evaluating the Hong Kong Benchmarks (Community) Pilot Program.

In our study, aimed at examining the effectiveness and impact of the Hong Kong Benchmarks (Community) Pilot Program, a career and life development (CLD) intervention program targeting non-engaged youth (NEY) in Hong Kong, we employed a pretest-posttest quasi-experimental design to compare changes in career-related competencies between a pilot group (N = 289) and a comparison group (N = 160). We also conducted five focus group interviews with the leaders of nongovernmental organizations, social workers, NEY, parents, and employers to explore the program's impacts on the CLD service provisions. Our quantitative results indicate that the piloting group showed greater improvement in two career-related competencies-youth career development competency and career and life development hope-than the comparison group. Meanwhile, our qualitative results suggest both the benefits and difficulties experienced by stakeholders in the program. The findings thus provide preliminary evidence of the Hong Kong Benchmarks (Community) Pilot Program's positive impacts on NEY and other important stakeholders. The implications of expanding the existing program and theorizing the community-based benchmark approach are also discussed.

Integrated intracellular organization and its variations in human iPS cells.

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour3,4-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the 'wiring' of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.

High-quality read-based phasing of cystic fibrosis cohort informs genetic understanding of disease modification.

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. We sequenced 477 individuals with cystic fibrosis (CF) using linked-read sequencing, which display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, demonstrating its utility for understanding complex CF alleles. These are visualized in a Web app, CFTbaRcodes, that enables interactive exploration of CFTR haplotypes present in this cohort. We perform fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus, an intestinal obstruction at birth associated with more severe CF outcomes and pancreatic disease. A 20-kb deletion polymorphism and a PRSS2 missense variant p.Thr8Ile (rs62473563) are shown to independently contribute to meconium ileus risk (p = 0.0028, p = 0.011, respectively) and are PRSS2 pancreas eQTLs (p = 9.5 × 10-7 and p = 1.4 × 10-4, respectively), suggesting the mechanism by which these polymorphisms contribute to CF. The phase information from linked reads provides a putative causal explanation for variation at a CF-relevant locus, which also has implications for the genetic basis of non-CF pancreatitis, to which this locus has been reported to contribute.

Prognostic Model of COVID-19 Severity and Survival among Hospitalized Patients Using Machine Learning Techniques.

We conducted a statistical study and developed a machine learning model to triage COVID-19 patients affected during the height of the COVID-19 pandemic in Hong Kong based on their medical records and test results (features) collected during their hospitalization. The correlation between the values of these features is studied against discharge status and disease severity as a preliminary step to identify those features with a more pronounced effect on the patient outcome. Once identified, they constitute the inputs of four machine learning models, Decision Tree, Random Forest, Gradient and RUSBoosting, which predict both the Mortality and Severity associated with the disease. We test the accuracy of the models when the number of input features is varied, demonstrating their stability; i.e., the models are already highly predictive when run over a core set of (6) features. We show that Random Forest and Gradient Boosting classifiers are highly accurate in predicting patients' Mortality (average accuracy ∼99%) as well as categorize patients (average accuracy ∼91%) into four distinct risk classes (Severity of COVID-19 infection). Our methodical and broad approach combines statistical insights with various machine learning models, which paves the way forward in the AI-assisted triage and prognosis of COVID-19 cases, which is potentially generalizable to other seasonal flus.

Development and Validation of the Career and Life Development Hope (CLDH) Scale among Non-Engaged Youth in Hong Kong.

Increasing career and life development hope (CLDH) is critical for the career and life pursuits of non-engaged youths (NEY) who face various disadvantages in the school-to-work transition, especially considering current challenging labor market conditions and the impacts of the pandemic. Nevertheless, research that explores the assessment of CLDH among NEY is scarce. To address this gap, this study aimed to develop and validate a CLDH measurement instrument. A total of 1998 NEY aged 13-29 years in Hong Kong participated in our study. Exploratory factor analysis of the 20-item CLDH scale suggested a two-factor structure-career and life development pathways (CLDP) and career and life development agency (CLDA)-which accounted for 63.08% of the total variance. The confirmatory factor analysis results show a good model fit (CFI = 0.934, TLI = 0.926, RMSEA = 0.060, 90% CI [0.055, 0.065], SRMR = 0.042) and all the items significantly represented the corresponding sub-constructs. The results also demonstrate a satisfactory internal consistency for all subscales and the full scale (0.89-0.95). Sub-group consistency across subsamples categorized by gender, age, and years of residence in Hong Kong was indicated. Correlations between the CLDH scale and subscales with other career-related and social well-being outcomes (i.e., youth career development competency, career adaptability, civic engagement, social contribution, and social integration) showed good concurrent validity. Our results support that the CLDH scale is a valid and reliable tool for measuring NEY's hope for career and life development in the Hong Kong context. Theoretical and practical implications of the findings are also discussed.

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease.

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.

Exercise and Quality-of-Life Outcomes of Two Versus Three Weekly Sessions of Pulmonary Rehabilitation.

PURPOSE: This study aimed to assess whether there are differences in exercise or health-related quality-of-life (HRQoL) outcomes following twice-weekly supervised sessions of pulmonary rehabilitation (PR) compared with three times weekly over an 8-wk program in patients with chronic obstructive pulmonary disease (COPD). METHODS: We conducted a quasi-experimental, single-center observational study using 198 subjects who completed two supervised PR sessions (intervention group) compared with 208 historical controls who completed three weekly sessions. We assessed between-group differences in outcomes after balancing groups using inverse probability of treatment weighting (IPTW) of propensity scores, followed by regression adjustment. RESULTS: Both groups achieved clinically and statistically significant improvements in exercise and HRQoL following the PR program. After IPTW and regression adjustment, the intervention group had a lower post-PR 6-min walk time by 1.2: 95% CI, -12.9 to 10.5 m ( P = .84), compared with the control group. Although post-PR COPD Assessment Test (CAT) scores decreased in both groups, the intervention group had a higher post-PR CAT score by 1.5: 95% CI, 0.37 to 2.66 a.u. ( P = .01), compared with the control group. All other HRQoL measures failed to reach statistical significance. None of the between-group differences reached minimal clinically important differences for COPD. CONCLUSIONS: Our findings support current international guidelines for twice-weekly supervised PR sessions combined with unsupervised home exercise sessions. We conclude there is no disadvantage in running a PR program for patients with COPD using twice-weekly supervised sessions compared with three times weekly supervised sessions.

Cystic fibrosis-related diabetes onset can be predicted using biomarkers measured at birth.

PURPOSE: Cystic fibrosis (CF), caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR), affects multiple organs including the exocrine pancreas, which is a causal contributor to cystic fibrosis-related diabetes (CFRD). Untreated CFRD causes increased CF-related mortality whereas early detection can improve outcomes. METHODS: Using genetic and easily accessible clinical measures available at birth, we constructed a CFRD prediction model using the Canadian CF Gene Modifier Study (CGS; n = 1,958) and validated it in the French CF Gene Modifier Study (FGMS; n = 1,003). We investigated genetic variants shown to associate with CF disease severity across multiple organs in genome-wide association studies. RESULTS: The strongest predictors included sex, CFTR severity score, and several genetic variants including one annotated to PRSS1, which encodes cationic trypsinogen. The final model defined in the CGS shows excellent agreement when validated on the FGMS, and the risk classifier shows slightly better performance at predicting CFRD risk later in life in both studies. CONCLUSION: We demonstrated clinical utility by comparing CFRD prevalence rates between the top 10% of individuals with the highest risk and the bottom 10% with the lowest risk. A web-based application was developed to provide practitioners with patient-specific CFRD risk to guide CFRD monitoring and treatment.

Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to Fusarium graminearum in Wheat.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the Fusarium graminearum fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type F. graminearum as compared with a Δtri5 mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including TaNFXL1, by treating tissues with DON directly. Previous work indicates that the NFXL1 ortholog represses trichothecene-induced defense responses and bacterial resistance in Arabidopsis, but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced TaNFXL1 gene expression in a susceptible wheat genotype relative to the F. graminearum-resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that TaNFXL1 represses F. graminearum resistance. Together, this suggests that targeting the TaNFXL1 gene may help to develop disease resistance in cultivated wheat.

The effects of cycled inhaled aztreonam on the cystic fibrosis (CF) lung microbiome.

BACKGROUND: To improve clinical outcomes, cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infections are prescribed inhaled anti-pseudomonal antibiotics. Although, a diverse microbial community exists within CF airways, little is known about how the CF microbiota influences patient outcomes. We hypothesized that organisms within the CF microbiota are affected by inhaled-antibiotics and baseline microbiome may be used to predict therapeutic response. METHODS: Adults with chronic P. aeruginosa infection from four clinics were observed during a single 28-day on/off inhaled-aztreonam cycle. Patients performed serial sputum collection, CF-respiratory infection symptom scores (CRISS), and spirometry. Patients achieving a decrease of ≥2 CRISS by day 28 were categorized as subjective responders (SR). The airway microbiome was defined by Illumina MiSeq analysis of the 16S rRNA gene. RESULTS: Thirty-seven patients (median 37.4 years and FEV1 44% predicted) were enrolled. No significant cohort-wide changes in the microbiome were observed between on/off AZLI cycles in either alpha- or beta-diversity metrics. However, at an individual level shifts were apparent. Twenty-one patients (57%) were SR and fourteen patients did not subjectively respond. While alpha-diversity metrics did not associate with response, patients who did not subjectively respond had a higher abundance of Staphylococcus and Streptococcus, and lower abundance of Haemophilus. CONCLUSIONS: The CF microbiome is relatively resilient to AZLI perturbations. However, associated changes were observed at the individual patient level. The relative abundance of key "off-target" organisms associated with subjective improvements suggesting that the microbiome may be used as a tool to predict patient response - potentially improving outcomes.

Improving Access to Healthcare with On-Line Medical Appointment System.

Access to medical care is in many countries an obstacle to timely health care and new technological options for improving the access are not fully utilized. In this project Business Process Modelling and Notation (BPMN) is applied to obtain an efficient, flexible and low cost medical appointment system for a medium size medical centre.

The use of fructosamine in cystic fibrosis-related diabetes (CFRD) screening.

OBJECTIVE: To determine whether serum fructosamine correlates with glycemic control and clinical outcomes in patients being screened for cystic fibrosis-related diabetes (CFRD). METHODS: Fructosamine and percent predicted forced expiratory volume in 1s (FEV1) were measured in patients undergoing a 2h oral glucose tolerance test (OGTT) for CFRD screening. Fractional serum fructosamine (FSF) was calculated as fructosamine/total protein. RESULTS: FSF exhibited a positive correlation with 2h OGTT results (r2=0.3201, p=0.009), and ROC curve analysis suggested that FSF can identify patients with an abnormal OGTT (AUC=0.840, p=0.0002). FSF also exhibited a negative correlation with FEV1 (r2=0.3732, p=0.035). Patients with FSF≥3.70µmol/g had significantly lower FEV1 (median 47%) compared to those with FSF<3.70µmol/g (median 90%; p=0.015). CONCLUSIONS: FSF correlated with both OGTT results and FEV1, and reliably identified patients with abnormal OGTT results. This simple blood test shows potential as an effective tool in CFRD screening.

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities.

Vocational functioning in schizotypal and paranoid personality disorders.

Impaired vocational functioning is a hallmark of schizophrenia, but limited research has evaluated the relationships between work and schizophrenia-spectrum personality disorders, including schizotypal (SPD) and paranoid personality disorder (PPD). This study compared employment history and job characteristics of 174 individuals drawn from the community or clinic, based on four personality disorder groups: SPD Only, PPD Only, SPD+PPD, and No SPD or PPD. Symptoms and cognitive functioning were also assessed. Both PPD and/or SPD were associated with lower rates of current employment, and a history of having worked at less cognitively complex jobs than people without these disorders. Participants with PPD were less likely to have a history of competitive work for one year, whereas those with SPD tended to have worked at jobs involving lower levels of social contact, compared with those without these disorders. When the effects of symptoms and cognitive functioning were statistically controlled, PPD remained a significant predictor of work history, and SPD remained a significant predictor of social contact on the job. The findings suggest that impaired vocational functioning is an important characteristic of SPD and PPD.