Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Preclinical Screening of Splice-Switching Antisense Oligonucleotides in PDAC Organoids.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated splicing factors or genetic alterations in splicing-regulatory cis -elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids (PDOs) are a promising platform to recapitulate key aspects of disease states and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) transfection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) organoids. This optimized delivery method allows fast and efficient screening of ASOs that reverse oncogenic alternative splicing. In combination with advancements in chemical modifications and ASO-delivery strategies, this method has the potential to accelerate the discovery of anti-tumor ASO drugs that target pathological alternative splicing.

Complete Genome Sequence of Pseudomonas sp. Strain NC02, Isolated from Soil.

We report here the complete genome sequence of Pseudomonas sp. strain NC02, isolated from soil in eastern Massachusetts. We assembled PacBio reads into a single closed contig with 132× mean coverage and then polished this contig using Illumina MiSeq reads, yielding a 6,890,566-bp sequence with 61.1% GC content.

Detection of levamisole exposure in cocaine users by liquid chromatography-tandem mass spectrometry.

Levamisole, a veterinary antihelminthic, was recently recognized as an adulterant in cocaine and is known to cause severe adverse reactions in some cocaine users. Because of the health concerns involving levamisole-adulterated cocaine, we developed a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of levamisole in urine. This method was used to determine the prevalence of levamisole in cocaine-positive patient samples. All cocaine-positive urine samples that were sent to the San Francisco General Hospital Clinical Laboratory were tested for levamisole for one month. For LC, an Agilent 1200 series was used with a C(18) column and a gradient of mobile phase A (0.05% formic acid) and B (acetonitrile/methanol). Detection was carried out with an Applied Biosystems QTRAP(®) LC-MS-MS. The levamisole LC-MS-MS method was linear over the range of 5-2500 ng/mL (r > 0.996). Interassay and intraassay CVs were < 6%. The lower limit of detection for levamisole was 0.5 ng/mL. Out of 949 total urine drug screens, 20% were positive for benzoylecgonine, and of those, 88% were positive for levamisole. The high prevalence of levamisole-adulterated cocaine and potential toxicity in cocaine users is a serious public health concern. These findings validate the utility of an LC-MS-MS method for the detection of levamisole.

Prevalence and correlates of opiate overdose among young injection drug users in a large U.S. city.

OBJECTIVES: The current study examines the prevalence and correlates of witnessing and experiencing opiate overdoses among a sample of young, injection drug users (IDUs) and non-injection drug users (NIDUs) in Baltimore, MD. METHODS: Data were derived from a longitudinal study of 15-30 year old IDUs and NIDUs (N=309) who had initiated heroin, cocaine, and/or crack use within 5 years prior to study enrollment. Chi-square and Wilcoxon rank-sum tests were used in bivariate analyses of demographic and drug use variables with each of the two dependent variables. Multivariate logistic regression models were used to identify correlates of experiencing and witnessing overdose. RESULTS: Twenty-nine percent of participants reported having ever experienced an opiate overdose and 57% reported having ever witnessed an overdose. Having ever experienced an opiate overdose was independently associated with being White (Adjusted Odds Ratio [AOR]=3.2; 95% Confidence Interval [CI]: 1.6, 6.4) recent homelessness (AOR=2.9; 95%CI: 1.5, 5.7); and length of injection, 5.6-6.9 years versus <5.6 years (AOR=4.0; 95%CI: 1.8-8.9); injecting 7.0-7.9 years versus <5.6 years (AOR=2.5; 95%CI: 1.03-6.1); injecting >8 versus <5.6 years (AOR=4.7; 95%CI: 2.2-10.2). Having witnessed an opiate overdose was independently associated with being White (AOR=2.4; 95%CI: 1.4, 4.1) and injecting >8 years versus <5.6 years (AOR=2.2; 95%CI: 1.2, 4.0). CONCLUSIONS: This study documents the high prevalence of witnessing and experiencing opiate overdoses among young, newly initiated IDUs and NIDUs. The results could inform the growing number of overdose prevention efforts throughout the U.S.