A Constructivist Grounded Theory Study on Mental Health Recovery from a Lived Experience Perspective in Singapore.

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.

The fibronectin ED-A domain enhances recruitment of latent TGF-ß-binding protein-1 to the fibroblast matrix.

Dysregulated secretion and extracellular activation of TGF-ß1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-ß-binding protein-1 (LTBP-1) and thus stores TGF-ß1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-ß1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-ß1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.

Predicting and Understanding the Enzymatic Inhibition of Human Peroxiredoxin 5 by 4-Substituted Pyrocatechols by Combining Funnel Metadynamics, Solution NMR, and Steady-State Kinetics.

Funnel metadynamics is a kind of computational simulation used to enhance the sampling of protein-ligand binding events in solution. By characterization of the binding interaction events, an estimated absolute binding free energy can be calculated. Nuclear magnetic resonance and funnel metadynamics were used to evaluate the binding of pyrocatechol derivatives (catechol, 4-methylcatechol, and 4-tert-butylcatechol) to human peroxiredoxin 5. Human peroxiredoxins are peroxidases involved in cellular peroxide homeostasis. Recently, overexpressed or suppressed peroxiredoxin levels have been linked to various diseases. Here, the catechol derivatives were found to be inhibitors against human peroxiredoxin 5 through a partial mixed type noncompetitive mechanism. Funnel metadynamics provided a microscopic model for interpreting the inhibition mechanism. Correlations were observed between the inhibition constants and the absolute binding free energy. Overall, this study showcases the fact that funnel metadynamics simulations can be employed as a preliminary approach to gain an in-depth understanding of potential enzyme inhibitors.

Robust liquid-infused surfaces through patterned wettability.

Liquid-infused surfaces display advantageous properties that are normally associated with conventional gas-cushioned superhydrophobic surfaces. However, the surfaces can lose their novel properties if the infused liquid drains from the surface. We explore how drainage due to gravity or due to an external flow can be prevented through the use of chemical patterning. A small area of the overall surface is chemically treated to be preferentially wetted by the external fluid rather than the infused liquid. These sacrificial regions disrupt the continuity of the infused liquid, thereby preventing the liquid from draining from the texture. If the regions are patterned with the correct periodicity, drainage can be prevented entirely. The chemical patterns are created using spray-coating or deep-UV exposure, two facile techniques that are scalable to generate large-scale failure-resistant surfaces.

Altered interactions between cardiac myosin binding protein-C and α-cardiac actin variants associated with cardiomyopathies.

The two genes most commonly associated with mutations linked to hypertrophic or dilated cardiomyopathies are ß-myosin and cardiac myosin binding protein-C (cMyBP-C). Both of these proteins interact with cardiac actin (ACTC). Currently there are 16 ACTC variants that have been found in patients with HCM or DCM. While some of these ACTC variants exhibit protein instability or polymerization-deficiencies that might contribute to the development of disease, other changes could cause changes in protein-protein interactions between sarcomere proteins and ACTC. To test the hypothesis that changes in ACTC disrupt interactions with cMyBP-C, we examined the interactions between seven ACTC variants and the N-terminal C0C2 fragment of cMyBP-C. We found there was a significant decrease in binding affinity (increase in Kd values) for the A331P and Y166C variants of ACTC. These results suggest that a change in the ability of cMyBP-C to bind actin filaments containing these ACTC protein variants might contribute to the development of disease. These results also provide clues regarding the binding site of the C0C2 fragment of cMyBP-C on F-actin.

Drug-related problems associated with direct oral anticoagulants: an observational cross-sectional study of medical record review by pharmacists in a large teaching hospital.

Background: Prescribing DOACs presents with challenges in the elderly and patients with renal and hepatic impairment. To mitigate safety risks, pharmacists have a role in detection, prevention, and resolution of DOAC-associated drug-related problems (DRPs). Objectives: To identify the types of DOAC-associated DRPs in patients on DOAC therapy and factors that predispose patients to DOAC-associated DRPs. Methods: An observational cross-sectional study was conducted in SGH from January 1, 2017, to May 31, 2019, on patients prescribed with a DOAC (rivaroxaban, dabigatran, and apixaban). Data were electronically extracted for patient demographics, clinical characteristics, and details of DOAC-related DRPs identified by pharmacists. Matching of DRP group to non-DRP group at a ratio of 1:2 based on gender, race, and DOAC was performed. The DRP group included patients with detected DRPs while non-DRP group included patients without them. Descriptive analysis was used to summarize patient characteristics and types of DOAC-associated DRPs. In the matched population, conditional logistic regression was used to calculate unadjusted (UOR) and adjusted odds (AOR) ratio to detect association of DOAC-associated DRPs with age, renal function, ≥2 comorbidities, and DOAC indication (atrial fibrillation [AF] vs venous thromboembolism). Results: A total of 8432 patients prescribed DOACs were analyzed, which consisted of 827 (9.8%) and 7602 (90.2%) patients with DRPs and no DRPs, respectively. The top DOAC-associated DRP was inappropriate drug regimen (n = 487, 60.1%). After matching, 2403 patients were analyzed, consisting of 801 patients from DRP group and 1602 from non-DRP group. Factors associated with DOAC-associated DRPs were statistically significant for renal function at creatinine clearance (CrCl) of >30 to 50 mL/min/1.73 m2 (AOR: 1.42; 95% CI: 1.14-1.76; P = .002), 15 to 30 mL/min/1.73 m2 (OR: 1.94; 95% CI: 1.42-2.66; P < .001), and <15 mL/min/1.73m2 (OR: 2.35; 95% CI: 1.13-4.88; P = .022), respectively, compared with a CrCl of >50 mL/min/1.73 m2 and DOAC indication for AF (AOR: 1.84; 95% CI: 1.47-2.30; P < .001) compared with venous thromboembolism. Conclusion: Inappropriate drug regimen was the most common DOAC-associated DRP. Impaired renal function and patients with AF increased the likelihood of DOAC-associated DRPs.

Lived experiences of mental health conditions in Singapore: A constructivist grounded theory study.

BACKGROUND: Since the 1990s, the mental healthcare field has begun shifting to conceptualisations of personal mental health recovery, emphasising the heterogeneous nature of how people develop and overcome the difficulties associated with mental ill health. Despite three decades of research on the topic, most recovery-oriented studies have been conducted in predominantly Western cultures, lacking the necessary nuances when applied in Asian settings. AIMS: We sought to contribute to a growing body of research to fill this gap by exploring the experiences of people who experience mental ill-health in Singapore. METHOD: We adopted a constructivist grounded theory approach and interviewed 21 people who had been diagnosed as experiencing a mental health condition. RESULTS: The core category emerging from interview participant perspectives was a 'roller coaster ride of confusion'. This overarching category was made up of the following four sub-categories - 'not understanding what was happening', 'losing control over self', 'unpacking the root of challenges' and 'trying to make sense of the situation'. CONCLUSIONS: Taken together, the journey of a person experiencing mental health recovery in Singapore is filled with obstacles and uncertainty due to various social and cultural influences such as family pressures, the competitiveness of society and the high-pressure nature of Singapore's educational system. Future research needs to better understand if these are generalisable experiences, and interventions to mitigate their impact need to be explored. Given the strong societal influences, change will take time. Still, this study gives a voice to the lived experiences of people who face mental health challenges in Singapore in the hope that their insights may assist future generations in developing a more mentally healthy society.

A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.

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.

Chromatin Accessibility and Transcriptional Differences in Human Stem Cell-Derived Early-Stage Retinal Organoids.

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.

Single-cell analyses identify circulating anti-tumor CD8 T cells and markers for their enrichment.

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA and TCR sequencing to detect and characterize "tumor-matching" (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared with nonmatching T cells in blood and were less exhausted than matching cells in tumors. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome, we identified candidate cell surface markers for TM cells in mice and patients and validated NKG2D, CD39, and CX3CR1 in mice. These data show that the TCR can be used to identify tumor-relevant cells for characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.

Outcomes of pharmacist-provided medication review in collaborative care for adult Singaporeans receiving hemodialysis.

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.

Prestress in the extracellular matrix sensitizes latent TGF-ß1 for activation.

Integrin-mediated force application induces a conformational change in latent TGF-ß1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-ß1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-ß1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-ß1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-ß1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-ß1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.

Integrins αvß5 and αvß3 promote latent TGF-ß1 activation by human cardiac fibroblast contraction.

AIMS: Pathological tissue remodelling by myofibroblast contraction is a hallmark of cardiac fibrosis. Myofibroblasts differentiate from cardiac fibroblasts under the action of transforming growth factor-ß1 (TGF-ß1), which is secreted into the extracellular matrix as a large latent complex. Integrin-mediated traction forces activate TGF-ß1 by inducing a conformational change in the latent complex. The mesenchymal integrins αvß5 and αvß3 are expressed in the heart, but their role in the activation of TGF-ß1 remains elusive. Here, we test whether targeting αvß5 and αvß3 integrins reduces latent TGF-ß1 activation by cardiac fibroblasts with the goal to prevent the formation of α-smooth muscle actin (α-SMA)-expressing cardiac myofibroblasts and their contribution to fibrosis. METHODS AND RESULTS: Using a porcine model of induced right ventricular fibrosis and pro-fibrotic culture conditions, we show that integrins αvß5 and αvß3 are up-regulated in myofibroblast-enriched fibrotic lesions and differentiated cultured human cardiac myofibroblasts. Both integrins autonomously contribute to latent TGF-ß1 activation and myofibroblast differentiation, as demonstrated by function-blocking peptides and antibodies. Acute blocking of both integrins leads to significantly reduced TGF-ß1 activation by cardiac fibroblast contraction and loss of α-SMA expression, which is restored by adding active TGF-ß1. Manipulating integrin protein levels in overexpression and shRNA experiments reveals that both integrins can compensate for each other with respect to TGF-ß1 activation and induction of α-SMA expression. CONCLUSIONS: Integrins αvß5 and αvß3 both control myofibroblast differentiation by activating latent TGF-ß1. Pharmacological targeting of mesenchymal integrins is a possible strategy to selectively block TGF-ß1 activation by cardiac myofibroblasts and progression of fibrosis in the heart.

The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

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.

Subdomain location of mutations in cardiac actin correlate with type of functional change.

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.