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MicroRNAs (miRNAs) are a family of small, non-coding RNA species functioning as negative regulators of multiple target genes including tumour suppressor genes and oncogenes. Many miRNA gene loci are located within cancer-associated genomic regions. To identify potential new amplified oncogenic and/or deleted tumour suppressing miRNAs in lung cancer, we inferred miRNA gene dosage from high dimensional arrayCGH data. From miRBase v9.0 (http://microrna.sanger.ac.uk), 474 human miRNA genes were physically mapped to regions of chromosomal loss or gain identified from a high-resolution genome-wide arrayCGH study of 132 primary non-small cell lung cancers (NSCLCs) (a training set of 60 squamous cell carcinomas and 72 adenocarcinomas). MiRNAs were selected as candidates if their immediately flanking probes or host gene were deleted or amplified in at least 25% of primary tumours using both Analysis of Copy Errors algorithm and fold change (≥ ± 1.2) analyses. Using these criteria, 97 miRNAs mapped to regions of aberrant copy number. Analysis of three independent published lung cancer arrayCGH datasets confirmed that 22 of these miRNA loci showed directionally concordant copy number variation. MiR-218, encoded on 4p15.31 and 5q35.1 within two host genes (SLIT2 and SLIT3), in a region of copy number loss, was selected as a priority candidate for follow-up as it is reported as underexpressed in lung cancer. We confirmed decreased expression of mature miR-218 and its host genes by qRT-PCR in 39 NSCLCs relative to normal lung tissue. This downregulation of miR-218 was found to be associated with a history of cigarette smoking, but not human papilloma virus. Thus, we show for the first time that putative lung cancer-associated miRNAs can be identified from genome-wide arrayCGH datasets using a bioinformatics mapping approach, and report that miR-218 is a strong candidate tumour suppressing miRNA potentially involved in lung cancer.

Asbestos-related lung cancer accounts for 4-12% of lung cancers worldwide. We have previously identified ADAM28 as a putative oncogene involved in asbestos-related lung adenocarcinoma (ARLC-AC). We hypothesised that similarly gene expression profiling of asbestos-related lung squamous cell carcinomas (ARLC-SCC) may identify candidate oncogenes for ARLC-SCC. We undertook a microarray gene expression study in 56 subjects; 26 ARLC-SCC (defined as lung asbestos body (AB) counts >20AB/gram wet weight (gww) and 30 non-asbestos related lung squamous cell carcinoma (NARLC-SCC; no detectable lung asbestos bodies; 0AB/gww). Microarray and bioinformatics analysis identified six candidate genes differentially expressed between ARLC-SCC and NARLC-SCC based on statistical significance (p<0.001) and fold change (FC) of >2-fold. Two genes MS4A1 and CARD18, were technically replicated by qRT-PCR and showed consistent directional changes. As we also found MS4A1 to be overexpressed in ARLC-ACs, we selected this gene for biological validation in independent test sets (one internal, and one external dataset (2 primary tumor sets)). MS4A1 RNA expression dysregulation was validated in the external dataset but not in our internal dataset, likely due to the small sample size in the test set as immunohistochemical (IHC) staining for MS4A1 (CD20) showed that protein expression localized predominantly to stromal lymphocytes rather than tumor cells in ARLC-SCC. We conclude that differential expression of MS4A1 in this comparative gene expression study of ARLC-SCC versus NARLC-SCC is a stromal signal of uncertain significance, and an example of the rationale for tumor cell enrichment in preparation for gene expression studies where the aim is to identify markers of particular tumor phenotypes. Finally, our study failed to identify any strong gene candidates whose expression serves as a marker of asbestos etiology. Future research is required to determine the role of stromal lymphocyte MS4A1 dysregulation in pulmonary SCCs caused by asbestos.

Brain metastases are a major cause of melanoma-related mortality and morbidity. We undertook whole-exome sequencing of 50 tumours from patients undergoing surgical resection of brain metastases presenting as the first site of visceral disease spread and validated our findings in an independent dataset of 18 patients. Brain metastases had a similar driver mutational landscape to cutaneous melanomas in TCGA. However, KRAS was the most significantly enriched driver gene, with 4/50 (8%) of brain metastases harbouring non-synonymous mutations. Hotspot KRAS mutations were mutually exclusive from BRAF V600 , NRAS and HRAS mutations and were associated with a reduced overall survival from the resection of brain metastases (HR 10.01, p  = 0.001). Mutations in KRAS were clonal and concordant with extracranial disease, suggesting that these mutations are likely present within the primary. Our analyses suggest that KRAS mutations could help identify patients with primary melanoma at higher risk of brain metastases who may benefit from more intensive, protracted surveillance.

Background MicroRNAs (MiRNA) are small non-coding RNAs that regulate gene expression. The aim of this study was to identify miRNAs differentially expressed between mild and moderately emphysematous lung, as well as their functional target mRNAs. Resected lung from patients with COPD undergoing lung cancer surgery was profiled using miRNA (Agilent Human miRNA profiler G4470 V1.01) and mRNA (OperonV2.0) microarrays. Cells of lung origin (BEAS-2B and HFL1) were profiled using mRNA microarrays (Illumina HumanHT-12 V3) after in vitro manipulation. Results COPD patients had mean (SD) age 68 (6) years, FEV 1 72 (17)% predicted and gas transfer (KCO) 70 (10)% predicted. Five miRNAs ( miR-34c , miR-34b , miR-149 , miR-133a and miR-133b ) were significantly down-regulated in lung from patients with moderate compared to mild emphysema as defined by gas transfer ( p  < 0.01). In vitro upregulation of miR-34c in respiratory cells led to down-regulation of predicted target mRNAs, including SERPINE1 , MAP4K4 , ZNF3 , ALDOA and HNF4A . The fold change in ex-vivo expression of all five predicted target genes inversely correlated with that of miR-34c in emphysematous lung, but this relationship was strongest for SERPINE1 (p = 0.05). Conclusion Differences in miRNA expression are associated with emphysema severity in COPD patients. MiR-34c modulates expression of its putative target gene, SERPINE1 , in vitro in respiratory cell lines and ex vivo in emphysematous lung tissue.

IMPORTANCE: Brain metastases occur in 60% of patients with advanced melanoma and are a major cause of melanoma related mortality and morbidity. Although our understanding of the molecular alterations associated with melanoma progression is improving, there are currently no validated biomarkers which might help identify those patients at highest risk of developing brain metastases. OBJECTIVE: To examine the somatic mutational and copy-number landscape of brain metastases that develop as the isolated first visceral site of recurrence − 'early brain-metastasis' compared to extracranial melanoma metastases. DESIGN, SETTING AND PARTICIPANTS: Whole−exome sequencing of 50 tumors from patients undergoing surgical resection of one or more brain metastasis occurring as the first site of visceral relapse were identified from prospectively maintained databases in Sydney, Wellington, New York and Cambridge. Whole exome sequencing analyses allowed mutational profiles to be compared to cutaneous melanomas in The Cancer Genome Atlas (SKCM-TCGA; n=358) and the Memorial Sloan Kettering (SKCM-MSK-IMPACT; n=186) datasets. An external dataset comprising a further 18 patients with surgically resected early brain metastasis from two additional academic centers served as an independent validation cohort. MAIN OUTCOMES AND MEASURES: To assess the frequency of driver mutations in early brain metastasis and their influence on survival. RESULTS: In concordance with the landmark melanoma sequencing studies, we identified mutations in BRAF (21/50, 42%), NRAS (14/50, 28%) and NF1 (11/50, 22%) as the most frequently mutated melanoma driver genes. When compared to the mutational landscape of cutaneous melanomas in TCGA (SKCM-TCGA), KRAS was the most significantly enriched driver gene, with 5/50 (10%) of brain metastases harboring non-synonymous mutations, of which 4/5 (80%) were in the hotspot positions of codons 12 and 61. This was significantly higher than the corresponding frequency of KRAS-mutations within the entire SKCM-TCGA (2% (7/358), p=0.009, Fisher′s Exact Test) as well as the SKCM-MSK-IMPACT cohort (1.6% (3/186), p=0.016). Variants in KRAS were mutually exclusive from BRAFV600, NRAS and HRAS mutations and were associated with a significantly reduced overall survival from resection of brain metastasis (relative to KRAS-wild type brain metastases) in multivariate Cox proportional hazard models (HR 1.80, 95% CI 1.46-24.89, p=0.013). Mutations in KRAS were also clonal and concordant with extracranial disease, which suggests these mutations are present within the primary tumor. CONCLUSIONS AND RELEVANCE: Our analysis, the largest to date, suggests that early metastases to the brain (presenting as the first site of visceral relapse) are characterized by significant enrichment of hotspot KRAS mutations, potentially implicating constitutive RAS-driven cellular programs in neurotropic metastatic behavior in these cases. Based on these data, we suggest that screening for KRAS mutations might help identify those patients with primary melanoma at higher risk of brain metastases or poor survival, and could help inform future surveillance strategies.

During the COVID-19 pandemic, individuals depended on risk information to make decisions about everyday behaviors and public policy. Here, we assessed whether an interactive website influenced individuals’ risk tolerance to support public health goals. We collected data from 11,169 unique users who engaged with the online COVID-19 Event Risk Tool ( https://covid19risk.biosci.gatech.edu/ ) between 9/22/21 and 1/22/22. The website featured interactive elements, including a dynamic risk map, survey questions, and a risk quiz with accuracy feedback. After learning about the risk of COVID-19 exposure, participants reported being less willing to participate in events that could spread COVID-19, especially for high-risk large events. We also uncovered a bias in risk estimation: Participants tended to overestimate the risk of small events but underestimate the risk of large events. Importantly, even participants who voluntarily sought information about COVID risks tended to misestimate exposure risk, demonstrating the need for intervention. Participants from liberal-leaning counties were more likely to use the website tools and more responsive to feedback about risk misestimation, indicating that political partisanship influences how individuals seek and engage with COVID-19 information. Lastly, we explored temporal dynamics and found that user engagement and risk estimation fluctuated over the course of the Omicron variant outbreak. Overall, we report an effective large-scale method for communicating viral exposure risk; our findings are relevant to broader research on risk communication, epidemiological modeling, and risky decision-making.

Chronic conditions can lead to physical, cognitive and social decline; thus, increasing an individual's dependence on family who assist with activities of daily living. Interprofessional collaborative practice (IPCP), involving two or more health professionals working with the patient and their family, is one model of care for the high-quality management of individuals with chronic conditions in primary care. Nevertheless, family carers have reported a disconnect between themselves and healthcare providers in previous research. This study aimed to explore the experiences and perspectives of family carers for individuals with chronic conditions, regarding their involvement in IPCP. Aspects of constructivist grounded theory methodology were used. Family carers of individuals with chronic conditions were invited to participate in a one-on-one, semistructured interview about their experiences with IPCP in the care of their loved one. Interview transcripts were analysed using Charmaz's four-step iterative process: (1) line-by-line coding, (2) focused coding, (3) categorisation of codes and (4) potential theme and subtheme development with memo writing to support each phase of analysis. The research team collaborated on reflexivity exercises, the conceptualisation of categories and the development of themes. Constructivist data analysis of interviews (average 40 min) with 10 family carers resulted in two themes. (1) Stepping in for my loved one represents the notion that carers take on external roles on behalf of their loved ones (subthemes: working with interprofessional teams, supporting independence and learning as I go). (2) Taking on the carer role, represents the internal factors that influence the external roles described in theme 1 (subthemes: feeling obligated to be involved and changing relationship dynamics). This study outlines the external actions and internal influences on family carer involvement in an interprofessional team. The required knowledge and support to care for their loved ones is currently learned in an ad hoc manner, and carers' resources should be better promoted by health professionals. Additionally, the relationship dynamics between a carer and their loved one change as the carer becomes more involved in IPCP and influences how and the extent health professionals involve family carers. Carers were the study population involved in this qualitative study. Patient advocates who have chronic conditions, and are informal family carers, were involved in the creation and design of this study, including a review of the research question, participant information sheet and the interview guide.

Background The unpredictable and toxic nature of the unregulated drug supply poses overdose and other health risks for people who use drugs (PWUD). Drug checking services can reduce risks by identifying adulterants in individuals’ drug supplies. In the United States (U.S.), DCS are increasingly offered through community harm reduction services that provide evidence-based risk reduction services for PWUD, such as syringe services programs (SSPs). However, PWUD face several multi-level barriers to consistently utilizing DCS and engaging in post-DCS safer drug use behaviors (e.g., discard, use less, avoid using alone, etc.). Staff from a San Diego-based mobile SSP and researchers drew from Social Cognitive Theory and the Social Ecological Model to develop a motivational-interviewing adjunctive intervention (MI-CHANCE) led by peers with lived substance use experience to address multi-level barriers and promote DCS engagement and adoption of post-DCS safer drug use behaviors among PWUD. Methods We employ a hybrid type-1 effectiveness–implementation trial with a primary goal of determining effectiveness of the MI-CHANCE adjunctive intervention in increasing DCS utilization (primary behavioral outcome) and safer drug use behaviors (secondary behavioral outcome), leading to reduced overdose risk (primary health outcome) and HIV/HCV incidence (secondary health outcome). We will recruit, consent, conduct a baseline interview, and randomize 588 PWUD who used opioids or stimulants within the week prior to receive either MI-CHANCE or an attention-control standard-of-care condition. All participants will be offered DCS following exposure to the intervention or control condition. We will conduct six-month follow-up with participants over a 30-month period to collect outcomes and hypothesized predictors, mediators and potential confounders. Our secondary goal is to assess MI-CHANCE implementation considerations guided by the RE-AIM/PRISM framework via annual in-depth interviews over the course of the trial with SSP staff ( N  = 5) and participants ( N  = 20), focusing on acceptability and feasibility, and the potential for scalability via interviews with a broad sample of diverse U.S. SSPs ( N  = 20). Discussion This hybrid trial will advance crucial knowledge on the effectiveness and implementation of an adjunctive intervention to promote DCS engagement and safer drug use behaviors to reduce overdose risk among PWUD. Trial registration ClinicalTrials.gov, NCT06855836. Registered February 28, 2025. https://clinicaltrials.gov/study/NCT06855836?term=NCT06855836/rank=1 .

Abtract Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays. A novel approach using whole-genome CRISPR-Cas9 screen indicates that apoptosis is the most important mediator of cfDNA release.

Optogenetic studies of the peripheral nervous system and spinal cord are enabled by soft, wirelessly powered biocompatible devices. Optogenetics allows rapid, temporally specific control of neuronal activity by targeted expression and activation of light-sensitive proteins. Implementation typically requires remote light sources and fiber-optic delivery schemes that impose considerable physical constraints on natural behaviors. In this report we bypass these limitations using technologies that combine thin, mechanically soft neural interfaces with fully implantable, stretchable wireless radio power and control systems. The resulting devices achieve optogenetic modulation of the spinal cord and peripheral nervous system. This is demonstrated with two form factors; stretchable film appliqués that interface directly with peripheral nerves, and flexible filaments that insert into the narrow confines of the spinal epidural space. These soft, thin devices are minimally invasive, and histological tests suggest they can be used in chronic studies. We demonstrate the power of this technology by modulating peripheral and spinal pain circuitry, providing evidence for the potential widespread use of these devices in research and future clinical applications of optogenetics outside the brain.