United States marijuana legalization and opioid mortality trends before and during the first year of the COVID-19 pandemic.

BACKGROUND: To determine if marijuana legalization was associated with reduced opioid mortality. STUDY DESIGN: The United States (US) opioid mortality trend during the 2010-2019 decade was compared in states and District of Columbia (jurisdictions) that had implemented marijuana legalization with states that had not. Acceleration of opioid mortality during 2020, the first year of the coronavirus disease 2019 (COVID-19) pandemic, was also compared in recreational and medicinal-only legalizing jurisdictions. METHODS: Joinpoint methodology was applied to the Centers for Disease Control and Prevention WONDER data. Trends in legalizing jurisdictions were cumulative aggregates. RESULTS: The overall opioid and fentanyl death rates and the percentage of opioid deaths due to fentanyl increased more during 2010-2019 in jurisdictions that legalized marijuana than in those that did not (pairwise comparison p = 0.007, 0.05, and 0.006, respectively). By 2019, the all-opioid and fentanyl death rates were 44 and 50 percent greater in the legalizing than in the nonlegalizing jurisdictions, respectively. When the COVID-19 pandemic hit in 2020, jurisdictions that implemented recreational marijuana legalization before 2019 had significantly greater increases in both overall opioid and fentanyl death rates than jurisdictions with medicinal-only legalization. For all-opioids, the mean (95 percent confidence interval) 2019-to-2020 increases were 46.5 percent (36.6, 56.3 percent) and 29.1 percent (20.2, 37.9 percent), respectively (p = 0.02). For fentanyl, they were 115.6 percent (80.2, 151.6 percent) and 55.4 percent (31.6, 79.2 percent), respectively (p = 0.01). CONCLUSIONS: During the past decade, marijuana legalization in the US was associated at the jurisdiction level with a greater acceleration in opioid death rate. An even greater increase in opioid mortality occurred in recreational-legalizing jurisdictions with the onset of the COVID-19 pandemic. Marijuana legalization is correlated with worsening of the US opioid epidemic.

United States marijuana legalization and opioid mortality epidemic during 2010-2020 and pandemic implications.

BACKGROUND: The hypothesis that marijuana availability reduces opioid mortality merits more complete testing, especially in a country with the world's highest opioid death rate and 2nd highest cannabis-use-disorder prevalence. METHODS: The United States opioid mortality rate was compared in states and District of Columbia that had implemented marijuana legalization with states that had not, by applying joinpoint methodology to Centers for Disease Control and Prevention data. Variables included race/ethnicity and fentanyl-type opioids (fentanyls). RESULTS: After the same rates during 2010-2012, the opioid mortality rate increased more rapidly in marijuana-legalizing than non-legalizing jurisdictions (2010-2020 annual pairwise comparison p = 0.003 for all opioids and p = 0.0004 for fentanyls). During the past decade, all four major race/ethnicities in the U.S. had evidence for a statistically-significant greater increase in opioid mortality rates in legalizing than non-legalizing jurisdictions. Among legalizing jurisdictions, the greatest mortality rate increase for all opioids was in non-Hispanic blacks (27%/year, p = 0.0001) and for fentanyls in Hispanics (45%/year, p = 0.0000008). The greatest annual opioid mortality increase occurred in 2020, the first year of the COVID-19 pandemic, with non-Hispanic blacks having the greatest increase in legalizing vs. non-legalizing opioid-death-rate difference, from 32% higher in legalizing jurisdictions in 2019 to more than double in 2020. CONCLUSIONS: Instead of supporting the marijuana protection hypothesis, ecologic associations at the national level suggest that marijuana legalization has contributed to the U.S.'s opioid epidemic in all major races/ethnicities, and especially in blacks. If so, the increased use of marijuana during the 2020-2022 pandemic may thereby worsen the country's opioid crisis.

A Single-Step, High-Dose Selection Scheme Reveals Distinct Mechanisms of Acquired Resistance to Oncogenic Kinase Inhibition in Cancer Cells.

Despite the remarkable clinical efficacy demonstrated by molecularly targeted cancer therapeutics, the benefits are typically temporary due to the emergence of acquired drug resistance. This has spurred a massive effort by the cancer research community to identify mechanisms used by cancer cells to evade treatment. Among the various methodologies developed and employed to identify such mechanisms, the most commonly used approach has been to model acquired resistance by exposing cancer cells in culture to gradually increasing concentrations of drug over an extended period of time. Here, we employed a less commonly used variation on this approach, wherein resistant cells are selected by immediately exposing cancer cells to a continuous, high concentration of drug. Using this approach, we isolated clones representing three distinct mechanisms of resistance to inhibition of MET kinase activity from a single clonally derived cancer cell line. The emergent clones had acquired resistance through engagement of alternative receptor tyrosine kinases either through upregulation of FGF3 or HBEGF or increased MAPK signaling through an activating V600E mutation in BRAF. Importantly, these mechanisms were not identified using the conventional "ramp-up" approach in previous studies that employed the same cell line. These results suggest that the particular nature of the selection scheme employed in cell culture modeling studies can determine which potential resistance mechanisms are identified and which ones may be missed, highlighting the need for careful consideration of the specific approach used to model resistance in cultured cells. SIGNIFICANCE: Through modeling resistance to MET kinase inhibition in cultured cancer cells using single-step, high-dose selection, these findings highlight that the specific nature of the selection protocol impacts which resistance mechanisms are identified.See related commentary by Floros et al., p. 25.

Increasing illness among people out of labor market - A Danish register-based study.

In spite of decades of very active labor market policies, 25% of Denmark's population in the working ages are still out-of-work. The aim of this study was to investigate whether that is due to consistent or even increasing prevalence of ill health. For the period of 2002-2011, we investigated if i) the prevalence of four chronic diseases (cardiovascular disease, diabetes, cancer and mental disorders) among those out-of-work had changed, ii) the occurrence of new cases of those diseases were higher among those who were already out-of-work, or iii) if non-health-related benefits were disproportionately given to individuals recently diagnosed with a disease compared to those without disease. The study was register-based and comprised all Danish residents aged 20-60. During the study period, the prevalence of cardiovascular diseases and mental disorders increased among both employed and non-employed people. The increased prevalence for mental disorder was particularly high among people receiving means-tested benefits. Disease incidence was higher among people outside rather than inside the labor market, especially for mental disorders. Employed people with incident diseases had an unsurprisingly increased risk of leaving the labor market. However, a high proportion of people with incident mental disorders received low level means-tested benefits in the three years following this diagnosis, which is concerning. Men treated for mental disorders in 2006 had high excess probability of receiving a cash-benefit, OR = 4.83 (4.53-5.14) for the period 2007-2010. The estimates were similar for women.

A conserved ncRNA-binding protein recruits silencing factors to heterochromatin through an RNAi-independent mechanism.

Long noncoding RNAs (lncRNAs) can trigger repressive chromatin, but how they recruit silencing factors remains unclear. In Schizosaccharomyces pombe, heterochromatin assembly on transcribed noncoding pericentromeric repeats requires both RNAi and RNAi-independent mechanisms. In Saccharomyces cerevisiae, which lacks a repressive chromatin mark (H3K9me [methylated Lys9 on histone H3]), unstable ncRNAs are recognized by the RNA-binding protein Nrd1. We show that the S. pombe ortholog Seb1 is associated with pericentromeric lncRNAs. Individual mutation of dcr1+ (Dicer) or seb1+ results in equivalent partial reductions of pericentromeric H3K9me levels, but a double mutation eliminates this mark. Seb1 functions independently of RNAi by recruiting the NuRD (nucleosome remodeling and deacetylase)-related chromatin-modifying complex SHREC (Snf2-HDAC [histone deacetylase] repressor complex).

Single-stranded annealing induced by re-initiation of replication origins provides a novel and efficient mechanism for generating copy number expansion via non-allelic homologous recombination.

Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA) suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR). Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA) mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA-mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution.

Loss of DNA replication control is a potent inducer of gene amplification.

Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is inappropriately re-replicated, but the importance of this stringent control on genome stability has not been tested. Here we show that re-replication in Saccharomyces cerevisiae can strongly induce the initial step of gene amplification, increasing gene copy number from one to two or more. The resulting amplicons consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intrachromosomally arrayed at their endogenous locus in direct head-to-tail orientation. These re-replication-induced gene amplifications are mediated by nonallelic homologous recombination between the repetitive elements. We suggest that re-replication may be a contributor to gene copy number changes, which are important in fields such as cancer biology, evolution, and human genetics.

MicroRNA silencing through RISC recruitment of eIF6.

MicroRNAs (miRNAs) are a class of small RNAs that act post-transcriptionally to regulate messenger RNA stability and translation. To elucidate how miRNAs mediate their repressive effects, we performed biochemical and functional assays to identify new factors in the miRNA pathway. Here we show that human RISC (RNA-induced silencing complex) associates with a multiprotein complex containing MOV10--which is the homologue of Drosophila translational repressor Armitage--and proteins of the 60S ribosome subunit. Notably, this complex contains the anti-association factor eIF6 (also called ITGB4BP or p27BBP), a ribosome inhibitory protein known to prevent productive assembly of the 80S ribosome. Depletion of eIF6 in human cells specifically abrogates miRNA-mediated regulation of target protein and mRNA levels. Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of the endogenous LIN-14 and LIN-28 target protein and mRNA levels. These results uncover an evolutionarily conserved function of the ribosome anti-association factor eIF6 in miRNA-mediated post-transcriptional silencing.

Trapping of a nonpeptide ligand by the extracellular domains of the gonadotropin-releasing hormone receptor results in insurmountable antagonism.

Drugs that exhibit insurmountable antagonism are proposed to provide improved clinical efficacy through extended receptor blockade. Long-term suppression of the gonadotropin-releasing hormone receptor (GnRHR) is an important therapeutic approach for a number of sex hormone-dependent diseases. In this study, we describe the mechanism and structural components required for insurmountable activity of a GnRHR antagonist. TAK-013 behaves as an insurmountable antagonist at the human receptor (hGnRHR) but as a surmountable antagonist at the macaque receptor (mGnRHR). Mutation of the eight residues that differ between hGnRHR and mGnRHR identified Ser-203 and Leu-300 in extracellular loops (ECL) 2 and 3 of hGnRHR as essential for the insurmountability of TAK-013. Substitution of the corresponding residues in mGnRHR with Ser and Leu (mGnRHR-P203S/V300L) converts TAK-013 to an insurmountable antagonist. In addition, mutation of Met-24 to Leu in the amino terminus of hGnRHR also ablates the insurmountable antagonism of TAK-013. The mechanism of insurmountability of TAK-013 was determined to be governed by its rate of dissociation from the receptor. Although the association rates of TAK-013 to hGnRHR, mGnRHR, and mGnRHR-P203S/V300L do not differ, the dissociation rate half-life correlates closely with the degree of insurmountability observed (169, 9, and 55 min, respectively). Taken together, these data suggest a model of the GnRHR in which ECL2, ECL3, and the amino terminus engage with TAK-013 upon its binding to the transmembrane region of the receptor. These additional interactions form a "trap door" above TAK-013, restricting its dissociation and thus resulting in its insurmountability.

Disk entry: a complication of transforaminal epidural injection--a case report.

Correctly identifying the etiology of low back pain can be challenging. The importance of making an expedient, accurate diagnosis of lumbar radicular pain cannot be overemphasized, as proper treatment is based on the outcome. The application of fluoroscopically guided, contrast enhancement during spinal injections is commonly performed, but is not without complications. Intradiskal placement of contrast dye during lumbar transforaminal epidural injection is important to identify, as serious potential complications, such as diskitis, may occur. Additionally, postprocedure assessment may not be accurate if contrast placement is not correctly identified. This case report reviews the literature on complications of transforaminal steroid injection and presents the first documentation of intradiskal placement of contrast dye in the United States. A 72-year-old man presenting with a right L4 radiculopathy underwent a fluoroscopically guided, contrast-enhanced lumbar transforaminal epidural steroid injection and experienced intradiskal placement of contrast as a complication of this procedure. Although the patient was treated prophylactically and achieved good results, the need for fluoroscopy and contrast enhancement during performance of spinal injections is reemphasized.