Supplementary Material for: Lysophosphatidic Acid Induces Ligamentum Flavum Hypertrophy Through the LPAR1/Akt Pathway

<b><i>Background/Aims:</i></b> Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy <b><i>Methods:</i></b> We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in <i>in vivo</i> studies. <b><i>Results:</i></b> The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our <i>in vitro</i> findings were supported by our <i>in vivo</i> evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. <b><i>Conclusion:</i></b> LPA-LPAR1-Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy

Supplementary Material for: Improvement of Spinosad Production upon Utilization of Oils and Manipulation of β-Oxidation in a High-Producing <b><i>Saccharopolyspora spinosa</i></b> Strain

Spinosad, a member of polyketide-derived macrolides produced in the actinomycete <i>Saccharopolyspora spinosa</i>, has been developed as a broad-spectrum and effective insecticide. The β-oxidation pathway could be an important source of building blocks for the biosynthesis of spinosad, thus the effect of vegetable oils on the production of spinosad in a high-yield strain was investigated. The spinosad production increased significantly with the addition of strawberry seed oil (511.64 mg/L) and camellia oil (520.07 mg/L) compared to the control group without oil (285.76 mg/L) and soybean oil group (398.11 mg/L). It also revealed that the addition of oils would affect the expression of genes involved in fatty acid metabolism, precursor supply, and oxidative stress. The genetically engineered strain, in which <i>fadD1</i> and <i>fadE</i> genes of <i>Streptomyces coelicolor</i> were inserted, produced spinosad up to 784.72 mg/L in the medium containing camellia oil, while a higher spinosad production level (843.40 mg/L) was detected with the addition of 0.01 mM of thiourea

Supplementary Material for: Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in Streptomyces Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes

<p>Spinosyns are a group of macrolide insecticides produced by <i>Saccharopolyspora spinosa</i>. Although <i>S. spinosa</i> can be used for industrial-scale production of spinosyns, this might suffer from several limitations, mainly related to its long growth cycle, low fermentation biomass, and inefficient utilization of starch. It is crucial to generate a robust strain for further spinosyn production and the development of spinosyn derivatives. A BAC vector, containing the whole biosynthetic gene cluster for spinosyn (74 kb) and the elements required for conjugal transfer and site-specific integration, was introduced into different <i>Streptomyces</i> hosts in order to obtain heterologous spinosyn-producing strains. The exconjugants of different <i>Streptomyces</i> strains did not show spinosyn production unless the rhamnose biosynthesis genes from <i>S. spinosa</i> genomic DNA were present and expressed under the control of a strong constitutive <i>ermE</i>*<i>p</i> promoter. Using this heterologous expression system resulted in yields of 1 μg/mL and 1.5 μg/mL spinosyns in <i>Streptomyces coelicolor</i> and <i>Streptomyces lividans</i>, respectively. This report demonstrates spinosyn production in 2 <i>Streptomyces</i> strains and stresses the essential role of rhamnose in this process. This work also provides a potential alternative route for producing spinosyn analogs by means of genetic manipulation in the heterologous hosts.</p

Supplementary Material for: Patient-reported Outcomes in Young Adults with Myeloproliferative Neoplasms

Introduction Genetic landscape, disease characteristics and clinical outcomes of young adults with myeloproliferative neoplasms (MPNs) were reported. However, data on patient-reported outcomes (PROs) in young adults with MPNs were rare. Methods We conducted a multicenter, cross-sectional study to compared the PROs in respondents with thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) by age at survey, including the young group (18-40 years), middle-aged group (41-60 years), and elderly group (> 60 years). Results Of the 1664 respondents with MPNs, 349 (21.0%) were young including 244 (69.9%) with ET, 34 (9.7%) with PV and 71 (20.3%) with MF. In multivariate analyses the young groups with ET and MF were associated with the lowest MPN-10 scores among the 3 age groups; those with MF, highest proportion of reporting negative impact of disease and therapy on their daily life and work. The young groups with MPNs had the highest physical component summary scores, but the lowest mental component summary scores in those with ET. The young groups with MPNs were most concerned about fertility; those with ET, treatment-related adverse events and long-term efficacy of treatment. Conclusions We concluded that young adults with MPNs have different PROs compared with middle-aged and elderly patients