S ilencing of SCD and SREBP1 Genes by Short Hairpin RNAs in Chicken Hepatic Cells in Vitro

RNA interference by short hairpin RNAs (shRNAs) is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes involved in denovo lipid biosynthesis were designed upon considering parameters such as secondary structures of shRNAs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target), synthesized and cloned in pENTR/U6 entry vector to knockdown the expression of SCD and SREBP1 genes. After transfection of these shRNA constructs into the chicken embryonic hepatocytes, expressions of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes was observed in hepatocytes. The shRNAs against SCD gene showed the knock down efficiency ranged from 20.4% (shRNA5) to 74.2% (shRNA2). In case of SREBP1 gene, the shRNAs showed knock-down efficiency ranging from 26.8% (shRNA4) to 95.85% (shRNA1). The shRNAs against both the genes introduced in chicken hepatocyte cells did not show any significant impact on expression of immune response genes (IFNA and IFNB) in those cells. These results clearly demonstrated the successful down regulation of the expression of SCD and SREBP1 genes by the shRNA molecules against both the target genes under in vitro condition. It is concluded that the shRNA molecules against SCD and SREBP1 genes showed great potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cells.

Insulin-like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

Insulin-like growth factor-1 (IGF-1)-related signaling is associated with prostate cancer progression. Links were explored between IGF-1 and expression of integrin adhesion receptors to evaluate relevance for growth and migration. Androgen-resistant PC3 and DU145 and androgen-sensitive LNCaP and VCaP prostate cancer cells were stimulated with IGF-1 and tumor growth (all cell lines), adhesion and chemotaxis (PC3, DU145) were determined. Evaluation of Akt/mTOR-related proteins, focal adhesion kinase (FAK) and integrin α and β subtype expression followed. Akt knock-down was used to investigate its influence on integrin expression, while FAK blockade served to evaluate its influence on mTOR signaling. Integrin knock-down served to investigate its influence on tumor growth and chemotaxis. Stimulation with IGF-1 activated growth in PC3, DU145, and VCaP cells, and altered adhesion and chemotactic properties of DU145 and PC3 cells. This was associated with time-dependent alterations of the integrins α3, α5, αV, and β1, FAK phosphorylation and Akt/mTOR signaling. Integrin blockade or integrin knock-down in DU145 and PC3 cells altered tumor growth, adhesion, and chemotaxis. Akt knock-down (DU145 cells) cancelled the effect of IGF-1 on α3, α5, and αV integrins, whereas FAK blockade cancelled the effect of IGF-1 on mTOR signaling (DU145 cells). Prostate cancer growth and invasion are thus controlled by a fine-tuned network between IGF-1 driven integrin-FAK signaling and the Akt-mTOR pathway. Concerted targeting of integrin subtypes along with Akt-mTOR signaling could, therefore, open options to prevent progressive dissemination of prostate cancer.

LIX1 controls digestive mesenchyme-derived cell fate decision by regulating cristae organization in mitochondria

Limb Expression 1 (LIX1) is a master regulator of digestive mesenchymal progenitor and GastroIntestinal Stromal Tumor (GIST) cell proliferation by controlling the expression of the Hippo effectors YAP1/TAZ and KIT. However, the underlying mechanisms of these LIX1- mediated regulations and tumor promotion remain to be elucidated. Here, we report that LIX1 is S-palmitoylated on cysteine 84 and localized in mitochondria. LIX1 knock-down affects the mitochondrial ultrastructure, resulting in decreased respiration and mitochondrial reactive oxygen species production. This is sufficient to downregulate YAP1/TAZ and reprogram KIT- positive GIST cells towards the smooth muscle cell lineage with reduced proliferative and invasive capacities. Mechanistically, LIX1 knock-down impairs the stability of the mitochondrial proteins PHB2 and OPA1 that are found in complexes with mitochondrial- specific phospholipids and are required for cristae organization. Supplementation with unsaturated fatty acids counteracts the effects of LIX1 knock-down on mitochondrial morphology and ultrastructure, restores YAP1/TAZ signaling, and consequently KIT levels. Altogether, our findings demonstrate that LIX1 contributes to GIST aggressive potential by modulating YAP1/TAZ and KIT levels, a process that depends on mitochondrial remodeling. Our work brings new insights into the mechanisms that could be targeted in tumors in which YAP1 and TAZ are implicated.

Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.

PPAR-γ Promotes the Polarization of Rat Retinal Microglia To M2 Phenotype By Regulating the Expression of CD200-CD200R1 Under Hypoxia

Objective Based on recent reports, peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms instigate the anti-inflammatory ability of PPAR-γ in microglia have not been expounded. In the present study, we explored the molecular mechanisms of the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat microglial cells. Methods shRNA expressing lentivirus was used to knock down PPAR-γ and CD200 genes. The hypoxia-induced polarization markers release (M1: iNOS, IL-1β, IL-6 and TNF-α; M2: Arg-1, YM1, IL-4 and IL-10) was assessed by RT-PCR, while PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200 and CD200Rs) were monitored by western blot and RT-PCR. Results Hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. In addition, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, while sh-PPAR-γ (PPAR-γ knock-down) had just the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype. Conclusion Results demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via CD200-CD200R1 pathway.

Barrier knock-down weir as an alternative technology for irrigation

Sand bags are one of the technologies still widely used to elevate water level. The sand bags are arranged in order to dam the river flow and to irrigate agricultural land, especially during the dry season. In line with its utilization, water supply of this technology is still less effective, thus, alternative technology is needed. This study aimed to test Barrier Knock-Down as an alternative technology that can be assembled and stored. This study was a quantitative research conducted by testing the prototypes. The tests were carried out using a monitor instrument Diver, piezometer. The tests of comparison were conducted in 4 types: Type 1, it was an L shape filled with sand+water; Type 2 was I shape filled with sand+water; Type 3 was an I shape filled with water; Type 4 was in an L shape filled with water. From the results of the study, type 1 is the best method for weir irrigation technology because of its ability to resist vertical and horizontal force than other types. Type 1-an L shape filled with sand+water with the value of safety factor rolling stability 5,28 and shear stability 2,23. Experiment result a stable elevation value in weir, did not experience a shift.

Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.

Effect of Short-Term Heating on Bioefficacy of Deltamethrin-Coated Long-Lasting Insecticidal Nets

The authors recently reported that long-lasting insecticidal nets (LLINs) distributed in Papua New Guinea (PNG) between 2013 and 2019, exhibited severely diminished efficacy to knock down and kill susceptible Anopheles mosquitoes. This coincided with a rise in malaria observed in PNG since 2015. Here, the authors show that LLIN bioefficacy is increased by heating LLINs prior to WHO cone bioassays. Unused LLINs with low bioefficacy, delivered to PNG in 2019, were heated to 120°C for 5 minutes. Cone bioassays were performed before and at 1 hour, 7 days, and 30 days after heating. This led to a significant increase in 24-hour mortality (17–61%) and 60-minute knock down (31–72%). The effect was sustained over 30 days. Bioassays are crucial in quality assurance of LLIN products. Our findings indicate that bioefficacy of LLINs can be increased by heating. This may have implications for quality assurance procedures used to assess LLINs.