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Introduction Liquid biopsies utilize cell-free DNA (cfDNA) in blood plasma or urine across various applications, including cancer detection, treatment selection, and treatment/minimal residual disease monitoring. Higher amplifiable copies recovered from liquid biopsies can lead to better cancer detection and monitoring. In this study, we evaluated the recovery of cfDNA amplifiable copies and circulating tumor DNA (ctDNA) dynamics using the nRichDX extraction method, which can extract cfDNA from a broad range of biological fluids (1mL - 20mL). cfDNA quality and ctDNA dynamics were assessed using Liquid IQ and UltraSEEK on the MassARRAY System. Methods Whole blood from healthy donors was collected in K2EDTA tubes, and plasma was isolated and then pooled in preparation for cfDNA extraction. Plasma aliquots at 5mL (6) and 20mL (6) were extracted using the Revolution Max20 cfDNA Extraction Kit. (12) replicate samples were extracted using QiAmp Circulating Nucleic Acid Kit. Half of the samples were spiked with a cfDNA standard containing a KRAS p.G12V mutation. The quantity and quality of extracted cfDNA were determined using the Liquid IQ panel on the MassARRAY system, which measures amplifiable cfDNA copies, high/low molecular weight dynamics, white blood cell (WBC) contamination along with a molecular barcode for sample matching. Recovery and frequency of the spiked ctDNA standard were performed with the UltraSEEK Lung V2 Panel, capable of detecting variants as low as 0.1% allele frequency. Results This study compared the amplifiable copies recovered from cfDNA extracted using the nRichDX and Qiagen kit. The amplifiable copies for the nRichDX and Qiagen methods, respectively, were: 5mL non-spiked 517+/-36 Vs. 398+/-17, 5mL spiked 946+/-99 Vs. 732+/-73; 20mL non-spiked 1526+/-26 Vs. 1071+/-55 and 20mL spiked 3004+/-278 Vs. 1777+/-207. All results were statistically different by conventional criteria, evaluated by a two-tailed t-test. Other cfDNA quality characteristics, such as low/high molecular weight DNA and WBC contamination, were concordant between the 2 methods. The spiked KRAS mutation was recovered consistently by both methods with a mutation frequency (5.3+/-0.3) and mutation significance z-score (296+/-19). Conclusion The nRichDX Revolution System extracts cfDNA and ctDNA with consistently high yields. Two sample T-Test analysis shows that nRichDX, at all levels, performed significantly better than Qiagen at p < 0.05. The results demonstrate the capability of the nRichDX system to recover more amplifiable copies with consistent cfDNA quality and ctDNA frequency and hence offer the opportunity to increase the sensitivity of detecting lower frequency mutations in cfDNA and a wider variety of biomarkers. Citation Format: Nafiseh Jafari, Mayer Saidian, Darryl Irwin. The nRichDX ® revolution sample prep system enables recovery of more amplifiable copies and more cancer fraction detection from cfDNA as evaluated on the MassARRAY ® System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6600.

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF-κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL-2, IL-6, IL-21, IL-23, tumor necrosis factor (TNF) α, and TNF-β were tested by enzyme-linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF-κB in MLs and the production of IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-α, and TNF-β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF-κB, IL-1β, IL-2, IL-6, IL-21, IL-23, TNF-α, and TNF-β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down-regulating protein levels of IL-1β and TNF-α, in which TLR4/MyD88/NF-κB signal pathway could be involved.

Cycloferin is an extract of the chemicals from the Cyclopia species, which grows only in small areas in the southwest and southeast of South Africa and has been consumed traditionally as a ...

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll‐like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF‐κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF‐κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL‐2, IL‐6, IL‐21, IL‐23, tumor necrosis factor (TNF) α, and TNF‐β were tested by enzyme‐linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF‐κB in MLs and the production of IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF‐κB, IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down‐regulating protein levels of IL‐1β and TNF‐α, in which TLR4/MyD88/NF‐κB signal pathway could be involved.

Objective: Accidental or intentional cyanide ingestion is an-ever present danger. Rapidly acting, safe, inexpensive oral cyanide antidotes are needed that can neutralize large gastrointestinal cyanide reservoirs. Since humans cannot be exposed to cyanide experimentally, we studied oral cyanide poisoning in rabbits, testing oral sodium thiosulfate with and without gastric alkalization. Setting: University research laboratory. Subjects: New Zealand white rabbits. Interventions: Seven animal groups studied; Groups 1-5 received high dose oral NaCN (50 mg, >LD100) and were treated immediately with oral (via nasogastric tube): 1) saline, 2) glycine, 3) sodium thiosulfate or 4) sodium thiosulfate and glycine, or 5) after 2 min with intramuscular injection of sodium nitrite and sodium thiosulfate plus oral sodium thiosulfate and glycine. Groups 6-7 received moderate dose oral NaCN (25 mg, LD70) and delayed intramuscular 6) saline or 7) sodium nitrite-sodium thiosulfate. Measurements and Main Results: All animals in the high dose NaCN group receiving oral saline or glycine died very rapidly, with a trend towards delayed death in glycine-treated animals; saline versus glycine-treated animals died at 10.3+3.9 and 14.6+5.9 min, respectively (p=0.13). In contrast, all sodium thiosulfate-treated high dose cyanide animals survived (p

Severe malnutrition resulting from anorexia nervosa or involuntary starvation leads to low weight, cognitive deficits, and increased mortality rates. In the present study, we examined whether fish oil supplementation, compared with canola oil, would ameliorate the morbidity and mortality associated with these conditions by normalizing endocannabinoid and monoaminergeric systems as well as other systems involved in satiety and cognitive function within the hypothalamus and hippocampus. Female Sabra mice restricted to 40% of their daily food intake exhibited decreased body weight, were sickly in appearance, displayed cognitive deficits, and had increased mortality rates. Strikingly, fish oil supplementation that contains high omega-3 fatty acids levels decreased mortality and morbidity, and normalized the expression of genes and neurotransmitters in the hippocampus and hypothalamus. Fish oil supplementation, but not canola oil, increased survival rates, improved general appearance, and prevented cognitive decline, despite the facts that both diets contained an equivalent number of calories and that there were no differences in weight between mice maintained on the two diets in 100% but decrease in the 40%. In the hypothalamus, the beneficial effects of fish oil supplementation were related to normalization of the endocannabinoid 2-arachidonylglycerol (2-AG), serotonin (5-HT) (p