Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms.

Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy.

The Lin28/let-7 axis regulates glucose metabolism.

The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating metabolism. When overexpressed in mice, both Lin28a and LIN28B promote an insulin-sensitized state that resists high-fat-diet induced diabetes. Conversely, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These phenomena occur, in part, through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. In addition, the mTOR inhibitor, rapamycin, abrogates Lin28a-mediated insulin sensitivity and enhanced glucose uptake. Moreover, let-7 targets are enriched for genes containing SNPs associated with type 2 diabetes and control of fasting glucose in human genome-wide association studies. These data establish the Lin28/let-7 pathway as a central regulator of mammalian glucose metabolism.

Positive Affect Processes and Posttraumatic Stress Disorder Symptoms: Findings from an Open Label and Uncontrolled Pilot Study using the Positive Memory Processing Technique.

Recently, a five-session Processing of Positive Memories Technique (PPMT) was proposed as a novel intervention for posttraumatic stress disorder (PTSD). One purported outcome of and mechanism underlying PPMT's effects on PTSD is improved positive affect processes. In this uncontrolled pilot study, we examined whether PPMT was associated with decreases in PTSD severity; and whether changes in positive affect levels, reactivity, and dysregulation related to changes in PTSD severity across sessions. The sample included 16 trauma-exposed participants seeking services at a University Psychology Clinic (Mage=27.44 years; 68.80% women). Multilevel linear growth models examined the main effects of each positive affect variable and their interactions with time on PTSD severity. PTSD severity decreased across PPMT treatment in each model (bs=-0.43 to -0.33; d=-0.03; ps<.001-0.008). There was a main effect of positive emotion dysregulation (b=1.16, d=0.11; p=0.009), but not of positive affect levels (p=0.821) or reactivity (p=0.356) on PTSD severity. However, positive affect processes did not modify the trajectory of PTSD severity across treatment. Regarding PTSD symptom clusters, there was an interaction between positive affect levels and time on alterations in arousal and reactivity (AAR) cluster severity (b=-0.01, p=0.036); individuals with positive affect levels 1 SD above the mean (b=-0.18, p<0.01) and at the mean (b=-0.10, p=0.01) had greater decreases in AAR cluster severity across treatment compared to individuals with positive affect levels 1 SD below the mean (b=-0.02, p=0.710). Findings suggest that PPMT may relate to improved PTSD symptoms; and that positive affect levels/dysregulation may be worthwhile targets for future investigations.

Artificial intelligence and clinical decision support: clinicians' perspectives on trust, trustworthiness, and liability.

Artificial intelligence (AI) could revolutionise health care, potentially improving clinician decision making and patient safety, and reducing the impact of workforce shortages. However, policymakers and regulators have concerns over whether AI and clinical decision support systems (CDSSs) are trusted by stakeholders, and indeed whether they are worthy of trust. Yet, what is meant by trust and trustworthiness is often implicit, and it may not be clear who or what is being trusted. We address these lacunae, focusing largely on the perspective(s) of clinicians on trust and trustworthiness in AI and CDSSs. Empirical studies suggest that clinicians' concerns about their use include the accuracy of advice given and potential legal liability if harm to a patient occurs. Onora O'Neill's conceptualisation of trust and trustworthiness provides the framework for our analysis, generating a productive understanding of clinicians' reported trust issues. Through unpacking these concepts, we gain greater clarity over the meaning ascribed to them by stakeholders; delimit the extent to which stakeholders are talking at cross purposes; and promote the continued utility of trust and trustworthiness as useful concepts in current debates around the use of AI and CDSSs.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Characterizing Patterns of Nurses' Daily Sleep Health: a Latent Profile Analysis.

BACKGROUND: Nursing is a demanding occupation characterized by dramatic sleep disruptions. Yet most studies on nurses' sleep treat sleep disturbances as a homogenous construct and do not use daily measures to address recall biases. Using person-centered analyses, we examined heterogeneity in nurses' daily sleep patterns in relation to psychological and physical health. METHODS: Nurses (N = 392; 92% female, mean age = 39.54 years) completed 14 daily sleep diaries to assess sleep duration, efficiency, quality, and nightmare severity, as well as measures of psychological functioning and a blood draw to assess inflammatory markers interleukin-6 (IL-6) and C-reactive protein (CRP). Using recommended fit indices and a 3-step approach, latent profile analysis was used to identify the best-fitting class solution. RESULTS: The best-fitting solution suggested three classes: (1) "Poor Overall Sleep" (11.2%), (2) "Nightmares Only" (8.4%), (3) "Good Overall Sleep" (80.4%). Compared to nurses in the Good Overall Sleep class, nurses in the Poor Overall Sleep or Nightmares Only classes were more likely to be shift workers and had greater stress, PTSD symptoms, depression, anxiety, and insomnia severity. In multivariate models, every one-unit increase in insomnia severity and IL-6 was associated with a 33% and a 21% increase in the odds of being in the Poor Overall Sleep compared to the Good Overall Sleep class, respectively. CONCLUSION: Nurses with more severe and diverse sleep disturbances experience worse health and may be in greatest need of sleep-related and other clinical interventions.

Factors associated with the use of the prescription monitoring program by prescribers and pharmacists in Texas.

PURPOSE: To describe Texas Prescription Monitoring Program (PMP) use and identify predictors of PMP query for opioid and benzodiazepine prescriptions by prescribers and pharmacists. METHODS: Dispensation and query records from the Texas PMP for opioid and benzodiazepine medications dispensed between October 1, 2016 and December 31, 2018 were linked using common patient identifiers. Autoregressive linear regression was used to assess trends in utilization. Hierarchical logistic models were specified to identify factors associated with provider and pharmacist query of opioid and benzodiazepine prescriptions. RESULTS: Despite a significant increase in the total number of pharmacists (ß = 169.85, p < 0.0001) and prescribers (ß = 301.59, p < 0.0001) who used the PMP every month, the ratio of active to registered pharmacists (ß = -0.0001, p = 0.75) and prescribers (ß = -0.0015, p = 0.10) did not change. Pharmacists and prescribers were significantly more likely to query opioid and benzodiazepine prescriptions of 14 days or more, and those issued to patients new to their practice. Pharmacists were most likely to query opioid prescriptions for oxycodone (aOR = 4.51, 95%CI = 4.42-4.60) and prescribers were most likely to query prescriptions for buprenorphine (aOR = 2.24, 95%CI = 2.15-2.35) compared to codeine. CONCLUSION: Changes in PMP utilization between October 2016 and December 2018 were driven by increasing registration, not increasing frequency of use among registered users. Use of the PMP is inconsistent and dependent upon patient characteristics thus limiting the utility of the PMP as a decision support tool. These results support the need for policy mandating PMP use in Texas and provide a useful baseline and framework to evaluate the effectiveness of mandate implementation.

Clinical decision making involving prescription drug monitoring programs: A factorial, vignette-based study among student pharmacists.

OBJECTIVES: Prescription drug monitoring programs (PDMPs) are state-maintained databases that providers may reference when deciding to prescribe or dispense controlled substances. As more states begin to mandate PDMP use at the point of care, it is imperative to assess how pharmacists use PDMP information when determining whether to fill a controlled substance prescription (CSP). The objective of this study was to evaluate which factors affected fourth-year student pharmacists' decision to fill an opioid prescription, their level of confidence in their decision making, and familiarity with the PDMP. METHODS: We used a 24 factorial design to present a series of text-based vignettes to fourth-year student pharmacists. Each participant received 8 vignettes (5 randomly selected, 3 fixed), representing a hypothetical hydrocodone-acetaminophen combination prescription with varying levels of the following dichotomous factors: doctor shopping, dosage, pharmacy shopping, and concurrent benzodiazepine prescription. Participants were asked to decide whether or not they would fill each of the hypothetical prescriptions they received. A multilevel model was used to measure the association between each of the vignette factors, age, race, sex, experience with PDMP, and the decision to refuse to fill a prescription. Each vignette response served as an independent observation. RESULTS: A total of 87 participants yielded 696 vignette responses. Participants were significantly more likely to refuse to fill prescriptions with doctor shopping (adjusted odds ratio [aOR] 19.86 [95% CI 10.78-36.58]), pharmacy shopping (6.78 [4.13-11.12]), dosage (1.83 [1.16-2.90]), or if the student pharmacist was of female sex (1.73 [1.02-2.93]). Concomitant benzodiazepine use was not associated with a no-fill decision (1.45 [0.92-2.27]). CONCLUSION: This study reveals that student pharmacists' decision to fill a prescription is dependent on both prescription characteristics and a patient's CSP history. The importance of PDMP history cannot be downplayed and suggests that PDMP use may be effective in informing patient care decisions. Still, the variability in filling decision highlights the need to teach a formulaic approach to CSP dispensing in colleges of pharmacy.

Identification and quantitation of clinically relevant microbes in patient samples: Comparison of three k-mer based classifiers for speed, accuracy, and sensitivity.

Infections are a serious health concern worldwide, particularly in vulnerable populations such as the immunocompromised, elderly, and young. Advances in metagenomic sequencing availability, speed, and decreased cost offer the opportunity to supplement or even replace culture-based identification of pathogens with DNA sequence-based diagnostics. Adopting metagenomic analysis for clinical use requires that all aspects of the workflow are optimized and tested, including data analysis and computational time and resources. We tested the accuracy, sensitivity, and resource requirements of three top metagenomic taxonomic classifiers that use fast k-mer based algorithms: Centrifuge, CLARK, and KrakenUniq. Binary mixtures of bacteria showed all three reliably identified organisms down to 1% relative abundance, while only the relative abundance estimates of Centrifuge and CLARK were accurate. All three classifiers identified the organisms present in their default databases from a mock bacterial community of 20 organisms, but only Centrifuge had no false positives. In addition, Centrifuge required far less computational resources and time for analysis. Centrifuge analysis of metagenomes obtained from samples of VAP, infected DFUs, and FN showed Centrifuge identified pathogenic bacteria and one virus that were corroborated by culture or a clinical PCR assay. Importantly, in both diabetic foot ulcer patients, metagenomic sequencing identified pathogens 4-6 weeks before culture. Finally, we show that Centrifuge results were minimally affected by elimination of time-consuming read quality control and host screening steps.

Randomized Trial of Labor Induction in Women 35 Years of Age or Older.

BACKGROUND: The risk of antepartum stillbirth at term is higher among women 35 years of age or older than among younger women. Labor induction may reduce the risk of stillbirth, but it also may increase the risk of cesarean delivery, which already is common in this older age group. METHODS: We conducted a randomized, controlled trial involving primigravid women who were 35 years of age or older. Women were randomly assigned to labor induction between 39 weeks 0 days and 39 weeks 6 days of gestation or to expectant management (i.e., waiting until the spontaneous onset of labor or until the development of a medical problem that mandated induction). The primary outcome was cesarean delivery. The trial was not designed or powered to assess the effects of labor induction on stillbirth. RESULTS: A total of 619 women underwent randomization. In an intention-to-treat analysis, there were no significant between-group differences in the percentage of women who underwent a cesarean section (98 of 304 women in the induction group [32%] and 103 of 314 women in the expectant-management group [33%]; relative risk, 0.99; 95% confidence interval [CI], 0.87 to 1.14) or in the percentage of women who had a vaginal delivery with the use of forceps or vacuum (115 of 304 women [38%] and 104 of 314 women [33%], respectively; relative risk, 1.30; 95% CI, 0.96 to 1.77). There were no maternal or infant deaths and no significant between-group differences in the women's experience of childbirth or in the frequency of adverse maternal or neonatal outcomes. CONCLUSIONS: Among women of advanced maternal age, induction of labor at 39 weeks of gestation, as compared with expectant management, had no significant effect on the rate of cesarean section and no adverse short-term effects on maternal or neonatal outcomes. (Funded by the Research for Patient Benefit Programme of the National Institute for Health Research; Current Controlled Trials number, ISRCTN11517275.).

A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway.

The pluripotency factor Lin28 blocks the expression of let-7 microRNAs in undifferentiated cells during development, and functions as an oncogene in a subset of cancers. Lin28 binds to let-7 precursor (pre-let-7) RNAs and recruits 3' terminal uridylyl transferases to selectively inhibit let-7 biogenesis. Uridylated pre-let-7 is refractory to processing by Dicer, and is rapidly degraded by an unknown RNase. Here we identify Dis3l2 as the 3'-5' exonuclease responsible for the decay of uridylated pre-let-7 in mouse embryonic stem cells. Biochemical reconstitution assays show that 3' oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3' oligouridylation as an RNA decay signal for Dis3l2, and identifies the first physiological RNA substrate of this new exonuclease, which is mutated in the Perlman syndrome of fetal overgrowth and causes a predisposition to Wilms' tumour development.

Direct medical expenditures associated with Alzheimer's and related dementias (ADRD) in a nationally representative sample of older adults - an excess cost approach.

OBJECTIVE: To estimate the excess direct annual healthcare expenditures associated with Alzheimer's and related dementias(ADRD) among community-dwelling older adults in the United States. METHODS: This retrospective cross-sectional study compared the annual healthcare expenditures between elderly individuals aged 65 years and older with ADRD (n = 662) and without ADRD (n = 13,398) using data from the Medical Expenditure Panel Survey (MEPS) for the years 2007, 2009, 2011 and 2013. Adjusted total annual medical expenditures was estimated using generalized linear model with gamma distribution and log link in 2013 U.S. dollars. Adjusted inpatient, outpatient, emergency, home healthcare and prescription drug expenditures, were estimated using two-part logit-generalized linear regression models. RESULTS: The adjusted mean total healthcare expenditures were higher for the ADRD group as compared to the no ADRD group($14,508 vs. $10,096). Among those with ADRD, 34.3% of the expenditures were for home healthcare as compared to 4.4% among those without ADRD. Among users, the ADRD group had significantly higher home healthcare ($3,240 vs. $566) and prescription drug expenditures($3,471 vs. $2,471). There were no statistically significant differences in inpatient, emergency room and outpatient expenditures between the ADRD and no ADRD group. CONCLUSION: ADRD in U.S. community-dwelling elders is associated with significant financial burden, primarily driven by increased home healthcare use.

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction.

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNA(miR)) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNA(miR)s resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNA(miR)s for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.

Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4).

Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.

Rationale and Argument for Subunit Mohs Excision in Nasal Reconstruction.

BACKGROUND: Optimal aesthetic results are achieved when nasal defects after Mohs micrographic surgery (MMS) are reconstructed as entire nasal subunits. OBJECTIVE: To illustrate the importance of reconstructing the nose in entire subunits and explore the possibilities of expanding the principles of subunit reconstruction to the concept of subunit Mohs excision. METHODS: An 83-year-old man presented for MMS to excise 3 lesions on the nasal ala. The surgeons elected to excise and reconstruct the entire subunit. RESULTS: Excellent aesthetic and functional results were obtained. CONCLUSION: When a defect greater than 50% of a nasal subunit is encountered during MMS, immediate marginal control excision of the entire subunit can be performed with subsequent reconstruction. This technique ultimately has the potential to deliver a more aesthetically pleasing outcome and should be, at the very least, considered by all Mohs surgeons.

Toenail paronychia.

Paronychia is an inflammation of the tissues alongside the nail. It may be acute or chronic and can be seen in isolation or in association with an ingrowing toenail. Acute paronychial infections develop when a disruption occurs between the seal of the nail fold and the nail plate, providing a portal of entry for invading organisms. The treatment of paronychia associated with an ingrowing toenail is aimed at treating the causal toenail. In paronychia not associated with an ingrowing toenail, antibiotics may cure an early infection but surgical drainage of an abscess is often required. In this case, an intra-sulcal approach is preferable to a nail fold incision. Chronic paronychia is less common in the feet than in the hands. It is a form of contact dermatitis and is frequently non-infective, however the chronically irritated tissue may become secondarily colonised by fungi. A dermatology consultation should be obtained for suspected chronic paronychia. Patients with chronic paronychia that is unresponsive to standard treatment should be investigated for unusual causes, such as malignancy. An algorithm for the treatment of paronychia is presented in this review.

A randomized trial of nicotine-replacement therapy patches in pregnancy.

BACKGROUND: Nicotine-replacement therapy is effective for smoking cessation outside pregnancy and its use is widely recommended during pregnancy. We investigated the efficacy and safety of nicotine patches during pregnancy. METHODS: We recruited participants from seven hospitals in England who were 16 to 50 years of age with pregnancies of 12 to 24 weeks' gestation and who smoked five or more cigarettes per day. Participants received behavioral cessation support and were randomly assigned to 8 weeks of treatment with active nicotine patches (15 mg per 16 hours) or matched placebo patches. The primary outcome was abstinence from the date of smoking cessation until delivery, as validated by measurement of exhaled carbon monoxide or salivary cotinine. Safety was assessed by monitoring for adverse pregnancy and birth outcomes. RESULTS: Of 1050 participants, 521 were randomly assigned to nicotine-replacement therapy and 529 to placebo. There was no significant difference in the rate of abstinence from the quit date until delivery between the nicotine-replacement and placebo groups (9.4% and 7.6%, respectively; unadjusted odds ratio with nicotine-replacement therapy, 1.26; 95% confidence interval, 0.82 to 1.96), although the rate was higher at 1 month in the nicotine-replacement group than in the placebo group (21.3% vs. 11.7%). Compliance was low; only 7.2% of women assigned to nicotine-replacement therapy and 2.8% assigned to placebo used patches for more than 1 month. Rates of adverse pregnancy and birth outcomes were similar in the two groups. CONCLUSIONS: Adding a nicotine patch (15 mg per 16 hours) to behavioral cessation support for women who smoked during pregnancy did not significantly increase the rate of abstinence from smoking until delivery or the risk of adverse pregnancy or birth outcomes. However, low compliance rates substantially limited the assessment of safety. (Funded by the National Institute for Health Research Health Technology Assessment Programme; Current Controlled Trials number, ISRCTN07249128.).