Accumulating evidence has revealed that dental anxiety (DA), as a dispositional factor toward the dental situation, is associated with the state anxiety (SA) and pain related to dental procedures. ...However, conclusions from individual studies may be limited by the treatment procedures that patients received, the tools used to assess DA, or the treatment stages when anxiety or pain was assessed. It is unclear whether DA, at the study level, accounts for the variance in pretreatment SA. The impact of DA and SA on pain at different treatment stages has not been systematically investigated. To address these questions, we present novel meta-analytical evidence from 35 articles (encompassing 47 clinical groups) that investigated DA in a clinical group. Subgroup analyses revealed that the studies of surgical and nonsurgical procedures did not significantly differ in either DA or pretreatment SA. Furthermore, metaregressions revealed DA as a significant predictor that explained the variance in SA assessed before and during treatment but not after treatment. The findings suggest that patient DA has a significant impact on patient SA. Metaregressions revealed DA as a significant predictor that explained the variance in expected pain, pain during treatment and posttreatment pain. In contrast, pretreatment SA was a significant predictor that explained the variance in expected pain. The findings reveal that DA has a consistent impact on pain through the entire period of dental treatment. Altogether, the findings highlight the role of DA as an overall indicator for anxiety and pain, across different types of dental procedures or treatment stages. We conclude that anxiety should be assessed as a critical step not only in anxiety management for high-DA patients, but also in pain control for all dental patients.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Onpattro, the first RNAi-based therapeutic to receive FDA approval, is enabled by a lipid nanoparticle (LNP) system that facilitates siRNA delivery into the cytoplasm of target cells (hepatocytes) ...following intravenous (i.v.) administration. These LNP-siRNA systems consist of four lipid components (ionizable cationic lipid, distearolyphosphatidycholine or DSPC, cholesterol, and PEG-lipid) and siRNA. The ionizable cationic lipid has been optimised for RNA encapsulation and intracellular delivery, and the PEG-lipids have been engineered to regulate LNP size and transfection potency. The roles of the other "helper" lipids, DSPC and cholesterol, remain less clear. Here we show that in empty LNP systems that do not contain siRNA, DSPC-cholesterol resides in outer layers, whereas in loaded systems a portion of the DSPC-cholesterol is internalised together with siRNA. It is concluded that the presence of internalised helper lipid is vital to the stable encapsulation of siRNA in the LNP and thus to LNP-siRNA function.
In empty LNP formulations, DSPC-cholesterol resides in outer layers, whereas in loaded systems some of the DSPC-cholesterol is internalized together with siRNA.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high degree of inflammation and profound immune suppression. Here we identify Yes-associated protein (Yap) as a critical regulator of the ...immunosuppressive microenvironment in both mouse and human PDAC. Within Kras:p53 mutant pancreatic ductal cells, Yap drives the expression and secretion of multiple cytokines/chemokines, which in turn promote the differentiation and accumulation of myeloid-derived suppressor cells (MDSCs) both in vitro and in vivo. Pancreas-specific knockout of Yap or antibody-mediated depletion of MDSCs promoted macrophage reprogramming, reactivation of T cells, apoptosis of Kras mutant neoplastic ductal cells and pancreatic regeneration after acute pancreatitis. In primary human PDAC, YAP expression levels strongly correlate with an MDSC gene signature, and high expression of YAP or MDSC-related genes predicts decreased survival in PDAC patients. These results reveal multifaceted roles of YAP in PDAC pathogenesis and underscore its promise as a therapeutic target for this deadly disease.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Lipid nanoparticles (LNP) can provide a clinically effective method for delivering small interfering RNA (siRNA) to silence pathological genes in hepatocytes. The gene silencing potency of these ...LNP-siRNA systems has been shown to depend on a variety of factors including association with serum factors such as ApoE and the pKa of component ionizable lipids. Here we investigate the influence of LNP size, an important parameter affecting tissue penetration of LNP systems, on the pharmacokinetics, biodistribution, and hepatic gene silencing potency of LNP-siRNA systems following intravenous administration. For LNP systems stabilized by a polyethylene glycol (PEG)-lipid that can dissociate from the LNP following injection, it is shown that small (diameter≤30nm) systems are considerably less potent than their larger counterparts. This is attributed in part to the ability of other lipid components, particularly the ionizable amino-lipid, to dissociate from the LNP following dissociation of the PEG-lipid. Small LNP stabilized by PEG-lipids with slow dissociation rates exhibited much reduced amino-lipid dissociation rates, however such systems are relatively impotent due to the continued presence of the PEG coating. These results demonstrate the delicate balance between the in vivo potency of LNP-siRNA systems and the residence times of component lipids in the LNP particle itself and suggest new directions to optimize the in vivo gene silencing potency of small LNP-siRNA systems.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Liposomes containing ionizable cationic lipids have been widely used for the delivery of nucleic acids such as small-interfering RNA and mRNA. The utility of cationic lipids with a permanent positive ...charge, however, is limited to in vitro transfection of cultured cells due to its dose-limiting toxic side effects observed in animals. Several reports have suggested that the permanently charged cationic lipids induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. We therefore hypothesized that the concomitant use of ROS inhibitor could reduce toxicity and improve drug efficacy. In this study, suppression of the cationic toxicity was evaluated using an ROS scavenger, edaravone, which is a low-molecular-weight antioxidant drug clinically approved for acute-phase cerebral infarction and amyotrophic lateral sclerosis. Cell viability assay in the mouse macrophage-like cell line RAW264 indicated that the concomitant use of edaravone were not able to suppress the cytotoxicity induced by cationic liposomes comprised of monovalent cationic lipid N-(1-2,3-dioleyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA) over a short period of time. Cationic lipids-induced necrosis was assumed to be involved in the cytotoxicity upon short-term exposure to cationic liposomes. On the other hand, the significant improvement of cell viability was observed when the short treatment with cationic liposomes was followed by exposure to edaravone for 24 h. It was also confirmed that apoptosis inhibition by ROS elimination might have contributed to this effect. These results suggest the utility of continuous administration with edaravone as concomitant drug for suppression of adverse reactions in therapeutic treatment using cationic liposomes.
Lipid nanoparticles (LNPs) containing short interfering RNA (LNP-siRNA) and optimized ionizable cationic lipids are now clinically validated systems for silencing disease-causing genes in hepatocytes ...following intravenous administration. However, the mechanism of formation and certain structural features of LNP-siRNA remain obscure. These systems are formed from lipid mixtures (cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid) dissolved in ethanol that is rapidly mixed with siRNA in aqueous buffer at a pH (pH 4) where the ionizable lipid is positively charged. The resulting dispersion is then dialyzed against a normal saline buffer to remove residual ethanol and raise the pH to 7.4 (above the pK a of the cationic lipid) to produce the finished LNP-siRNA systems. Here we provide cryogenic transmission electron microscopy (cryo-TEM) and X-ray evidence that the complexes formed between siRNA and ionizable lipid at pH 4 correspond to tightly packed bilayer structures with siRNA sandwiched between closely apposed monolayers. Further, it is shown that ionizable lipid not complexed to siRNA promotes formation of very small vesicular structures at pH 4 that coalesce to form larger LNP structures with amorphous electron dense cores at pH 7.4. A mechanism of formation of LNP-siRNA systems is proposed whereby siRNA is first sandwiched between closely apposed lipid monolayers at pH 4 and subsequently trapped in these structures as the pH is raised to 7.4, whereas ionizable lipid not interacting with siRNA moves from bilayer structure to adopt an amorphous oil phase located in the center of the LNP as the pH is raised. This model is discussed in terms of previous hypotheses and potential relevance to the design of LNP-siRNA systems.
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IJS, KILJ, NUK, PNG, UL, UM
The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue ...maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.
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•AMPK blocks Shh-induced transcriptional activity•AMPK reduces GLI1 protein level and stability•AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074•GLI13A protein is resistant to AMPK and has higher stability and oncogenic ability
Li. et al. show that AMPK is linked to the Hh signaling pathway. Activation of AMPK phosphorylates GLI1, a Hedgehog transcriptional activator, and inhibits Hh activity. GLI1 phosphorylation decreases GLI1 protein stability and reduces cell growth, colony formation, and tumor growth in mice.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for ...SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Coronavirus disease 2019 is a public health challenge requiring rapid testing for the detection of infections and transmission. Nucleic acid amplification tests targeting SARS coronavirus 2 (CoV2) ...are used to detect CoV2 in clinical samples. Real-time reverse transcription quantitative PCR is the standard nucleic acid amplification test for CoV2, although reverse transcription loop-mediated isothermal amplification is used in diagnostics. The authors demonstrate a sequence-specific reverse transcription loop-mediated isothermal amplification-based nucleic acid amplification assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay using labeled primers and a quencher oligonucleotide. This assay can achieve rapid (30 min) and sensitive (1000 plaque-forming units/ml) fluorescence detection of CoV2 (WA1/2020), B.1.1.7 (Alpha) and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.
The authors describe a sequence-specific nucleic acid amplification assay (fluorescence-quenched reverse transcription loop-mediated isothermal amplification) based on a modified reverse transcription loop-mediated isothermal amplification assay that utilizes a fluorescence-labeled reporter primer and a short complementary oligonucleotide quencher to detect SARS coronavirus 2 in minimally processed clinical nasal swab samples. The fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay is completed in 30 min without purifying RNA and achieves reproducible, sensitive and specific (1000 plaque-forming units/ml) detection of SARS coronavirus 2 WA1/2020 and three SARS coronavirus 2 variant viruses while not signaling on three closely related human coronaviruses or two other heterologous human respiratory viruses.
Lipid nanoparticles (LNPs) containing short-interfering RNA (LNP-siRNA systems) are a promising approach for silencing disease-causing genes in hepatocytes following intravenous administration. ...LNP-siRNA systems are generated by rapid mixing of lipids in ethanol with siRNA in aqueous buffer (pH 4.0) where the ionizable lipid is positively charged, followed by dialysis to remove ethanol and to raise the pH to 7.4. Ionizable cationic lipids are the critical excipient in LNP systems as they drive entrapment and intracellular delivery. A recent study on the formation of LNP-siRNA systems suggested that ionizable cationic lipids segregate from other lipid components upon charge neutralization to form an amorphous oil droplet in the core of LNPs. This leads to a decrease in intervesicle electrostatic repulsion, thereby engendering fusion of small vesicles to form final LNPs of increased size. In this study, we prepared LNP-siRNA systems containing four lipid components (hydrogenated soy phosphatidylcholine, cholesterol, PEG-lipid, and 1,2-dioleoyl-3-dimethylammonium propane) by microfluidic mixing. The effects of preparation parameters lipid concentration, flow rate ratio (FRR), and total flow rate, dialysis process, and complex formation between siRNA and ionizable cationic lipids on the physicochemical properties siRNA entrapment on the particle size and polydispersity index (PDI) were investigated using a design of experiments approach. The results for the preparation parameters showed no impact on siRNA encapsulation, but lipid concentration and FRR significantly affected the particle size and PDI. In addition, the effect of FRR on the particle size was suppressed in the presence of anionic polymers such as siRNA as compared to the case of LNPs alone. More intriguingly, unlike empty LNPs, a decrease in the PDI and an increase in the particle size occurred after dialysis in the LNP-siRNA systems. Such changes by dialysis were suppressed at FRR = 1. These findings provide useful information to guide the development and manufacturing conditions for LNP-siRNA systems.
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IJS, KILJ, NUK, PNG, UL, UM
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