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Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.

Determining the molecular mechanisms that lead to the development of heart failure will help us gain better insight into the most costly health problem in the Western world. To understand the roles that the actin protein plays in the development of heart failure, we have taken a systematic approach toward characterizing human cardiac actin mutants that have been associated with either hypertrophic or dilated cardiomyopathy. Seven known cardiac actin mutants were expressed in a baculovirus system, and their intrinsic properties were studied. In general, the changes to the properties of the actin proteins themselves were subtle. The R312H variant exhibited reduced stability, with a T(m) of 53.6 °C compared to 56.8 °C for WT actin, accompanied with increased polymerization critical concentration and Pi release rate, and a marked increase in nucleotide release rates. Substitution of methionine for leucine at amino acid 305 showed no impact on the stability, nucleotide release rates, or DNase-I inhibition ability of the actin monomer; however, during polymerization, a 2-fold increase in Pi release was observed. Increases to both the T(m) and DNase-I inhibition activity suggested interactions between E99K actin molecules under monomer-promoting conditions. Y166C actin had a higher critical concentration resulting in a lower Pi release rate due to reduced filament-forming potential. The locations of mutations on the ACTC protein correlated with the molecular effects; in general, mutations in subdomain 3 affected the stability of the ACTC protein or affect the polymerization of actin filaments, while mutations in subdomains 1 and 4 more likely affect protein-protein interactions.

Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.

Many patients with melanoma treated with immune checkpoint inhibitors (ICIs) do not derive response. Preclinical and retrospective studies identified that inhibition of the cyclooxygenase (COX) pathway may improve response to ICI treatment. This prospective single site phase II trial accrued patients with advanced/metastatic melanoma. Participants underwent high-dose aspirin daily combined with pembrolizumab and ipilimumab every 3 weeks for 4 cycles followed by high-dose aspirin and pembrolizumab monotherapy. The primary endpoint was objective response rate (ORR). Longitudinal sampling of blood was performed to assess peripheral immune correlates. Twenty-seven subjects were enrolled with median follow-up of 32 months. An ORR of 62.9% was reached prior to discontinuation due to low likelihood of achieving the pre-specified ORR of 80%. 17 patients (63%) experienced a treatment-related adverse event (TRAEs) grade 3 or higher. A per-protocol analysis showed that patients able to continue aspirin alongside ICI through the induction period experienced significant survival benefit. Ten cytokines and increased regulatory T cells in the periphery correlated with beneficial response. The addition of high-dose aspirin to combination ICI within this study results in response comparable to ICI alone. Future clinical studies of COX inhibition will need to focus on mitigation of AEs to establish the clinical utility of this combination.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

Background Patients receiving hemodialysis are predisposed to drug related problems (DRPs). While collaborative care (CC) models with pharmacist involvement can reduce DRP occurrence, few have examined its impact on clinical and economic outcomes. Objective To determine whether a CC model with pharmacist-provided medication review can reduce unplanned admissions and healthcare utilization in patients receiving hemodialysis, compared to usual care (UC). Setting Outpatient nephrology clinic of a tertiary hospital in Singapore. Method In this retrospective observational study, patients who were taking more than 10 medications or had prior unplanned admissions were included. Patients were identified as being managed under CC (n = 134) if they received comprehensive pharmacist-provided review, or under the UC (n = 190) if they did not. Those perceived to be at greater risk were given priority for receiving CC. All outcomes analyses were adjusted for covariates. Main outcome measure The primary outcome was incidence of unplanned admissions within 6 months post index visit. Secondary outcomes included length of stay (LOS), mortality and healthcare utilization cost. Results CC reduced unplanned admissions by 27% (IRR 0.73, 95% CI 0.54–0.99, p  = 0.047) and shortened mean LOS by 1.3 days [6.7 (2.6) vs. 8.0 (3.2), p  < 0.001] compared to UC. There were no significant differences in mortality ( p  = 0.189) or mean healthcare utilization cost ( p  = 0.165) between groups. Pharmacists identified 515 DRPs with 429 (83.3%) resolved after review. Conclusion The CC model with pharmacist-provided medication review reduced unplanned admissions and LOS in patients receiving hemodialysis. Further studies are warranted to confirm reductions in mortality and healthcare utilization.

Purpose Contemporary interpretations of mental health recovery emphasize the need to understand better the lived experience of people with mental health conditions and how they define the idea of recovery. While traction for such ideas has been building in many western countries, the recovery movement is still in its early stages within most Asian countries. Hence, this study aims to add to this growing effort by understanding various factors that influence the recovery of people with mental health conditions in Singapore. Design/methodology/approach Braun and Clarke’s thematic analysis was used on the qualitative data provided by 21 participants. Findings Four themes were revealed: double-edged nature of social support, challenges accessing mental health support, personalized coping strategies and societal influences. As a result, the authors could better understand various factors that influenced the recovery process of people with mental health conditions in Singapore, such as the high costs and low levels of literacy regarding pathways to mental health services. In addition, the importance of societal influences on mental health recovery was highlighted, and their impact could be seen through the participants’ sharing. Research limitations/implications These findings form a foundation from which future research and recovery-oriented interventions can work from to provide more tailored approaches to supporting people with mental health conditions. Originality/value This study identified cultural nuances to notions of recovery that are commonly found in personal recovery constructs that need to be considered when working within culturally diverse populations.

More contemporary personal recovery conceptualisation of mental health recovery emphasize the need to consider the perspectives of people who experience mental ill-health. Most lived experience research has been done in Western cultures with relatively few studies in Asian ones, creating a gap that needs to be addressed due to differences in cultural worldviews. This study explores the notion of recovery from the lens of people experiencing mental health challenges in Singapore. We adopted a constructivist grounded theory perspective to evaluate qualitative data from 21 participants. The core category which best represented what recovery meant was “reconciling and living with experiences of mental ill-health”. Our findings suggest that a variety of societal aspects greatly influence perceptions of mental health recovery in Singapore, as participants often shared their desire to live a meaningful life within society but could only do so if they found a way to manage their symptoms more effectively.

Aim: To assess trainee’s knowledge and practice behavior changes with the revised basic MMS training program. Methods: Kirkpatrick’s four-level evaluation model was adopted in this study. A questionnaire was used to seek trainees’ feedback on the training program. For learning progression, trainees’ self- perception of their competency pre- and post-training, and results from a multiple-choice questions (MCQ) test were assessed. Thirty-eight behavioral changes in the patient care delivery and problem solving competency cluster of the General Level Competency Framework (GLF) were compared pre- and 6 months post-training. Evaluation of level-4 results is pending as advanced training is in progress. Results: Fourteen out of fifteen trainees who had completed the basic MMS training responded to the online questionnaire. Eleven trainees (78.6%) rated basic MMS training program as good or excellent. Positive response was highest in tutorials (84.3%), followed by continuous education sessions (70.6%) and clinic attachments (67.6%). Suggestions for improvement include increasing the frequency and opportunities of hands-on patient interview at clinic attachments. Sixty-four percent of trainees perceived improvement (p<0.013) in delivering patient care post-training (median=3; IQR: 3-4) than pre-training (median=3; IQR: 2.75-3). All but one trainee passed the MCQ test (median score: 80; IQR: 76-86). A greater proportion of patient care delivery and problem solving behaviors (69.2% and 35.9% respectively) in GLF was graded highest performance at post-training compared to pre-training (35.3% and 14.3% respectively). Conclusions: The revised training program is well-received by trainees and feedback from trainees provides insights into future improvements in the content and delivery of the program.

This dissertation analyzes the differences between private and non-private firms in two contexts. Chapters 1 and 2 examine the electricity industry in the United States and the motivation behind electric utilities' usage of demand side management programs. The first chapter focuses on load management programs, which decrease electricity demand during the peak hours of the day. It looks into the impact of a plausibly exogenous decrease in natural gas prices on the utilization and capacity of these programs. The second chapter analyzes the relationship between electricity market deregulation and electric utilities' energy efficiency activity. The third chapter investigates the impact of Chinese enterprise restructuring on employment, wage bills, and productivity. All three chapters show that different objectives due to ownership type lead to differences in firm behavior.