Determinants of environmental degradation: Evidenced-based insights from ASEAN economies.

This paper examines the effect of per capita income, trade openness, energy consumption, and financial development on the ASEAN region's environmental degradation. We obtained data from the World Development Indicators (WDI) during the years 2001-2020 to test our research model. For analysis, we applied panel data techniques such as pooled least squares, fixed effects, generalized least squares (GLS), and two stages least squares (2SLS). We also conducted the causality analysis to determine the direction of the relationship among the models' variables. Our models' results show the validity of the environmental Kuznets curve (EKC) hypothesis in the ASEAN region as per capita income and its square term possess positive and negative coefficients. The results also show the presence of an N-shaped EKC in ASEAN region a deviation from the conventional literature. Moreover, the results illustrate that increasing energy consumption, trade openness, and financial development positively contribute to environmental degradation in ASEAN economies. The causality analysis shows a two-way causality between trade openness and financial development, and environmental degradation and trade openness. We observed oneway causality, running a) from energy consumption and per capita income towards financial development, b) from per capita income towards trade openness, and c) from financial development towards environmental degradation. The study's results contribute to the environmental degradation literature and provide a better understanding of environmental degradation for policymakers in ASEAN economies.

Pre-treatment predictors of response for assessing outcomes to standard treatment in infection with HCV genotype 3.

OBJECTIVE: To determine the role of pre-treatment predictors of response in assessing outcomes to standard treatment in HCV genotype 3. STUDY DESIGN: Observational study. PLACE AND DURATION OF STUDY: Department of Medicine, KRL General Hospital, Islamabad, from December 2004 to December 2006. METHODOLOGY: All patients with positive anti-HCV and PCR genotype 3a were recruited and written and informed consent was taken. Patients were treated with standard Interferon plus Ribavirin therapy (IFN alpha-2a, 3MU t.i.w 24 weeks plus Ribavirin 1000-1200 mg/day) for 6 months. The effect of pre-treatment factors influencing outcome i.e. age, gender, weight, baseline ALT, necroinflammatory grade, fibrosis and steatosis on the final outcome were further analyzed by univariate logistic regression analysis. RESULTS: Response rates to standard Interferon plus Ribazole therapy were studied in 190 patients. The end-of-treatment complete response (EOTCR) was seen in 81% (n=155) of the patients, whereas 17% (n=33) were non-responders (NR). Sustained viral response (SVR) was seen in 58% (n=112) patients and 24% (n=45) were relapsers. SVR was higher in patients without steatosis (OR=2.52, 95% CI=1.356- 4.71, p=0.04). Higher SVR was seen in patients weighing less than 65 kg, as compared with weight>65 kg (OR=2.277, 95% CI=1.246-4.161, p=0.007). The other variables were not found to be significantly associated with improved SVRs. CONCLUSION: Out of the studied predictors, body weight and presence of steatosis, were statistically related to treatment outcome. Pre-treatment host factors can predict response to treatment that can help in individualizing treatment and patient selection and optimize treatment outcomes.

Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle Function and Dystrophin Restoration in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. OBJECTIVE: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. METHODS: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. RESULTS: A chronic, monthly regimen of high dose PMO increased dystrophin rescue in mdx mice and improved specific force in the extensor digitorum longus (EDL) muscle. However, monthly high dose PMO administration still results in variable dystrophin expression localized throughout various muscles. CONCLUSIONS: High dose monthly PMO administration restores dystrophin expression and increases muscle force; however, the variability of dystrophin expression at both the inter-and intramuscular level remains. Additional strategies to optimize PMO uptake including increased dosing frequencies or combination treatments with other yet-to-be-defined therapies may be necessary to achieve uniform dystrophin restoration and increases in muscle function.

Ultrasensitive Hybridization-Based ELISA Method for the Determination of Phosphorodiamidate Morpholino Oligonucleotides in Biological samples.

Determining the concentration of oligonucleotide in biological samples such as tissue lysate and serum is essential for determining the biodistribution and pharmacokinetic profile, respectively. ELISA-based assays have shown far greater sensitivities compared to other methods such as HPLC and LC/MS. Here, we describe a novel ultrasensitive hybridization-based ELISA method for quantitating morpholino oligonucleotides in mouse tissue lysate and serum samples. The assay has a linear detection range of 5-250 pM (R2 > 0.99).

Complex phenotypes associated with STIM1 mutations in both coiled coil and EF-hand domains.

Dominant mutations in STIM1 are a cause of three allelic conditions: tubular aggregate myopathy, Stormorken syndrome (a complex phenotype including myopathy, hyposplenism, hypocalcaemia and bleeding diathesis), and a platelet dysfunction disorder, York platelet syndrome. Previous reports have suggested a genotype-phenotype correlation with mutations in the N-terminal EF-hand domain associated with tubular aggregate myopathy, and a common mutation at p.R304W in a coiled coil domain associated with Stormorken syndrome. In this study individuals with STIM1 variants were identified by exome sequencing or STIM1 direct sequencing, and assessed for neuromuscular, haematological and biochemical evidence of the allelic disorders of STIM1. STIM1 mutations were investigated by fibroblast calcium imaging and 3D modelling. Six individuals with STIM1 mutations, including two novel mutations (c.262A>G (p.S88G) and c.911G>A (p.R304Q)), were identified. Extra-neuromuscular symptoms including thrombocytopenia, platelet dysfunction, hypocalcaemia or hyposplenism were present in 5/6 patients with mutations in both the EF-hand and CC domains. 3/6 patients had psychiatric disorders, not previously reported in STIM1 disease. Review of published STIM1 patients (n = 49) confirmed that neuromuscular symptoms are present in most patients. We conclude that the phenotype associated with activating STIM1 mutations frequently includes extra-neuromuscular features such as hypocalcaemia, hypo-/asplenia and platelet dysfunction regardless of mutation domain.

Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides.

Antisense oligonucleotide (AON)-induced exon skipping is one of the most promising strategies for treating Duchenne muscular dystrophy (DMD) and other rare monogenic conditions. Phosphorodiamidate morpholino oligonucleotides (PMOs) and 2'-O-methyl phosphorothioate (2'OMe) are two of the most advanced AONs in development. The next generation of peptide-conjugated PMO (P-PMO) is also showing great promise, but to advance these therapies it is essential to determine the pharmacokinetic and biodistribution (PK/BD) profile using a suitable method to detect AON levels in blood and tissue samples. An enzyme-linked immunosorbent assay (ELISA)-based method, which shows greater sensitivity than the liquid chromatography-mass spectrometry method, is the method of choice for 2'OMe detection in preclinical and clinical studies. However, no such assay has been developed for PMO/P-PMO detection, and we have, therefore, developed an ultrasensitive hybridization-based ELISA for this purpose. The assay has a linear detection range of 5-250 pM (R(2)>0.99) in mouse serum and tissue lysates. The sensitivity was sufficient for determining the 24-h PK/BD profile of PMO and P-PMO injected at standard doses (12.5 mg/kg) in mdx mice, the dystrophin-deficient mouse model for DMD. The assay demonstrated an accuracy approaching 100% with precision values under 12%. This provides a powerful cost-effective assay for the purpose of accelerating the development of these emerging therapeutic agents.

Elusive sources of variability of dystrophin rescue by exon skipping.

BACKGROUND: Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials. METHODS: In the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays. RESULTS: The triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression. CONCLUSIONS: While we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.