De novo gene synthesis by an antiviral reverse transcriptase.

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

Characterizing Point-of-Care Ultrasound Credentialing in Pediatric Emergency Departments.

OBJECTIVE: It is unclear which pediatric emergency departments (PEDs) have a point-of-care ultrasound (POCUS) credentialing process or if this process is consistent per expert guidelines. Our objective was to describe formalized POCUS credentialing processes across PEDs that are active in the pediatric emergency medicine POCUS (P2) Network. METHODS: A survey was developed from nationally recommended credentialing guidelines. This anonymous survey was sent out to the P2 Network comprising more than 230 members involved in pediatric POCUS. The survey was analyzed using descriptive analysis with counts and percentages. RESULTS: A total of 36 PEDs responded to the survey. All departments had a faculty member in charge of maintaining the credentialing process, and all faculty members had POCUS education available; 88.6% of education was scheduled didactics or bedside teaching. There were 80.6% of PEDs that had a process for internally credentialing faculty. Some PEDs offered protected education for POCUS, however, 44.8% had <50% of their faculty credentialed. There were 4 PEDs that offered incentives for completion of POCUS credentialing including salary bonuses; only 1 offered shift buy down as incentive. That PED had 100% of its faculty credentialed. All PEDs performed quality assurance on POCUS scans done in the ED, most done weekly. Billing for scans occurred in 26 PEDs. Skin/soft tissue and focused assessment with sonography for trauma were the 2 most common applications credentialed. CONCLUSIONS: Among PEDs surveyed, there was a lack of standardization of POCUS resources and components of credentialing. Incentives may be beneficial in improving credentialing faculty and standardizing the credentialing process.

Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera.

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. To bridge data obtained from animal sera with human sera, we analyzed neutralizing antibody titers in human and hamster single infection sera in a highly controlled setting using the same authentic virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity.

Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

Durability of CLOSE-Guided Pulmonary Vein Isolation in Persistent Atrial Fibrillation: A Prospective Remapping Study.

BACKGROUND: Recurrence of paroxysmal atrial fibrillation (AF) following pulmonary vein isolation (PVI) is presumably caused by pulmonary vein (PV) reconnections. However, there is little data available on the durability of PVI and incidence of arrhythmia recurrence in patients with persistent AF. OBJECTIVES: To evaluate the lesion durability by means of an a priori planned remapping procedure in patients with persistent AF undergoing CLOSE-guided PVI. METHODS: In a prospective study, we included patients with symptomatic, persistent AF undergoing CLOSE-guided radiofrequency ablation. Irrespective of AF recurrence, a redo procedure was mandated 6 months following the index procedure to evaluate PV reconnections. The outcome of AF ablation was based on clinical recurrence and 7-day Holter electrocardiogram 3 and 6 months after the index procedure and 3, 6, and 12 months after the redo procedure. RESULTS: Of 30 patients included, 26 (81% men; median age 68 years) underwent the planned remapping study a median of 6 months after the index procedure, whereas 4 patients without recurrence refused a repeat procedure. In total, 78 of 102 (76%) PVs showed durable isolation and 15 patients (58%) presented complete isolation of all PVs. Beyond the blanking period, 6 of 26 patients (23%) had arrhythmia recurrence before the redo procedure. Recurrence had occurred in 33% of patients with complete isolation of all veins and in 9% of patients with PV reconnections (P = 0.197). After re-PVI in patients with PV reconnections and additional ablation in patients with recurrence but durable PVI, 17 of 26 patients (65%) were free of arrhythmia after 12 months. CONCLUSIONS: In patients with persistent AF, CLOSE-guided PVI resulted in durable rate of PVI on a per-vein and per-patient level of 76% and 58%, respectively. Arrhythmia recurrence was numerically higher in patients with durable PVI compared with patients without.

A facile one step route that introduces functionality to polymer powders for laser sintering.

Laser Sintering (LS) is a type of Additive Manufacturing (AM) exploiting laser processing of polymeric particles to produce 3D objects. Because of its ease of processability and thermo-physical properties, polyamide-12 (PA-12) represents ~95% of the polymeric materials used in LS. This constrains the functionality of the items produced, including limited available colours. Moreover, PA-12 objects tend to biofoul in wet environments. Therefore, a key challenge is to develop an inexpensive route to introduce desirable functionality to PA-12. We report a facile, clean, and scalable approach to modification of PA-12, exploiting supercritical carbon dioxide (scCO2) and free radical polymerizations to yield functionalised PA-12 materials. These can be easily printed using commercial apparatus. We demonstrate the potential by creating coloured PA-12 materials and show that the same approach can be utilized to create anti-biofouling objects. Our approach to functionalise materials could open significant new applications for AM.

Exposure to pro-tobacco and anti-tobacco media messages and events and smoking behaviour among adolescents in Gambia.

BACKGROUND: Despite the widespread prevalence of adolescent smoking in Gambia, a West African country, there is limited research exploring the relationships between exposure to pro-tobacco and anti-tobacco media messages and events and smoking behaviour among young people. This study investigates the interplay of these exposures and smoking behaviour among 11-17-year-old adolescents in Gambia. METHODS: Secondary data analysis was conducted using the 2017 Gambia Global Youth and Tobacco Survey (GYTS), which included a total of 9,127 respondents. Descriptive and inferential analyses, including proportions, Pearson's chi-squared tests, and multivariable logistic regression models, were employed to estimate adjusted odds ratios (aOR) with 95% confidence intervals (CI). RESULTS: The final model revealed significant associations between exposure to anti-tobacco media messages and events and smoking behaviour. Adolescents exposed to anti-tobacco media messages had a 29% increased odds of smoking (aOR 1.29,CI = 1.08,1.53) compared to those unexposed, while exposure to anti-tobacco media events showed a 31% increased odds (aOR 1.31,CI = 1.09,1.59) compared to those unexposed. Exposure to pro-tobacco messages, such as witnessing tobacco use on TV (aOR 1.41, CI = 1.17,1.69) and owning objects with tobacco brand logos (aOR 1.49,CI = 1.19,1.86), was associated with higher odds of smoking. Covariates, including sex, age, and exposure to smoking behaviour by significant others, also demonstrated associations with smoking behaviour. Notably, male respondents showed significantly higher odds of smoking (aOR = 4.01,CI = 3.28,4.89) compared to females. Respondents aged 15 years and older had increased odds of smoking (aOR = 1.47,CI = 1.22,1.76) compared to those below 15 years old. Those whose fathers smoke displayed higher odds of smoking (aOR = 1.35, CI = 1.04,1.76) compared to individuals with non-smoking parents. Additionally, those whose closest friends smoke showed remarkably higher odds of smoking (aOR = 2.87,CI = 2.37, 3.48) compared to those without such influence. CONCLUSION: This study underscores the significant impact of exposure to both anti-tobacco and pro-tobacco media messages and events on smoking behaviour among adolescents in Gambia. However, pro-tobacco messages had a greater influence on smoking prevalence than anti-tobacco messages and events. Understanding these associations is crucial for devising effective public health interventions aimed at reducing tobacco use in this population.

Neighborhood Light at Night and Noise Levels, and Long-Term Sleep Trajectories in the Southern Community Cohort Study.

While light at night (LAN) and noise levels have been linked to suboptimal sleep outcomes, little is known about the link between these factors and long-term suboptimal sleep trajectories. The current study examined the association of neighborhood LAN and nighttime noise with long-term sleep trajectories in a cohort of Black individuals and White individuals predominantly from low-income communities. We used data from the Southern Community Cohort Study (N = 28,759 Black individuals and 16,276 White individuals). Sleep duration was self-reported at baseline and after an average of five years of follow-up, based on which we constructed nine sleep trajectories: normal-normal (optimal, reference), short-short, long-long, short-long, long-short, normal-short, normal-long, short-normal, and long-normal. LAN and nighttime noise were derived from satellite imagery and model-based estimates, respectively. Multinomial logistic regression was used to determine the relationship between LAN and noise exposures and sleep trajectories. Higher exposures to LAN and nighttime noise were associated with multiple suboptimal long-term sleep trajectories. In the total sample, higher LAN was associated with higher odds of long-long (OR Q5 vs. Q1 = 1.23 (CI = 1.02, 1.48)) and long-short (OR = 1.35 (CI = 1.06, 1.72)) trajectories, while higher nighttime noise was associated with short-short (1.19 (1.07, 1.31)), long-short (1.31 (1.05, 1.64)), and normal-song (1.16 (1.01, 1.34)) trajectories. Black and White individual-specific results showed qualitatively similar patterns between Black individuals and White individuals, although we also observed suggestive evidence for Black-White individual differences. In conclusion, elevated LAN and nighttime noise levels were associated with various suboptimal long-term sleep trajectories. However, it is noteworthy that the light and noise measures in our study may not accurately reflect individual-level exposures, and residual confounding from other factors is a concern. Future studies should use more accurate exposure measurements, collect information on and control for a wider range of factors, and examine whether reductions in neighborhood light and noise levels may contribute to improved long-term sleep health.

The Quantification of Muscle Activation During the Loaded Carry Movement Pattern.

The 'loaded carry' is a popular resistance training activity that activates core musculature across multiple movement planes while the body is in locomotion. 'Hold' exercises are similar to carry exercises but lack the locomotive aspect. Both carry and hold exercises can be completed bilaterally (farmer's carry (FC) and hold (FH)) or unilaterally (suitcase carry (SC) and hold (SH)). A deeper understanding of muscle activation between the FC and SC and intensity-matched FH and SH might improve their application. Healthy, college-aged individuals were recruited and surface electromyography of the rectus abdominis (RA), external oblique (EO), longissimus (LT), and multifidus (MF) was measured bilaterally using standard procedures. Participants completed time- and intensity-matched randomized sets of the plank, FC, SC, FH, and SH separated by 5-minute rests. A one-way ANOVA was utilized to compare exercises. The FC/FH load averaged 50.7±1.9 kg, where it was used across equally weighted dumbbells. The FC elicited higher activation bilaterally in the LT, MF, RA, and EO, compared to the FH. The SC/SH single-dumbbell load averaged 25.3±0.95 kg. There was greater activation bilaterally in the LT and MF during the SC compared to the SH. However, on the ipsilateral side of the SC, the RA and EO displayed greater activation compared to the SH, but this was not different on the contralateral side. The FC and SC were characterized by increased core muscle activation bilaterally, with the SC exhibiting unique additions to ipsilateral muscle activation.

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer.

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remains unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We find that ApbE flavinylation sites exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models.

The Epidemiology of Hospital-Treated Alopecia Areata in Denmark, 1995-2016.

INTRODUCTION: Alopecia areata (AA) is an autoimmune skin disease presenting as nonscarring hair loss. Information on the epidemiology of AA, especially the occurrence of AA and its subtypes within the general population, is scarce. The study aimed to estimate the incidence rates and prevalence of hospital-treated AA and its subtypes in Denmark and to examine the demographic and clinical characteristics of patients with AA, including comorbidities and use of prescription medications. METHODS: This was a cohort study based on data from administrative and health registers in Denmark in 1995-2016. The study included individuals who were (1) registered with a hospital inpatient or hospital-based outpatient clinic diagnosis of AA between 1995 and 2016 in the Danish National Patient Registry covering encounters at all Danish hospitals, (2) alive and resided in Denmark anytime between 1995 and 2016, (3) aged ≥ 12 years, and (4) resided uninterrupted in Denmark during the 12 months before the first AA diagnosis during the study period. RESULTS: During the study period, 2778 individuals with an incident hospital-based diagnosis of AA were identified; 63.1% were female and 28.7% of the patients were aged ≥ 50 years. Over the study period, the overall incidence rate for any hospital-treated AA per 100,000 person-years was 2.62 (95% confidence interval [CI], 2.53-2.72), and the overall prevalence in 2016 was 71.7 (95% CI 69.4-74.1) per 100,000 persons. Both incidence rate and prevalence increased over time. Prevalence of most hospital-treated comorbidities or history of medication use was below 10% and was similar in the alopecia totalis (AT)/alopecia universalis (AU) and non-AT/AU subtypes of AA. CONCLUSION: This cohort study reported incidence rates and prevalence over time and characteristics of individuals with hospital-treated AA in Denmark, which are in agreement with those previously reported in this population.

Pushing the Isotopic Envelope: When carrier channels pollute their neighbors' signals.

Individual cells are the foundational unit of biology, and understanding their functions and interactions is critical to advancing our understanding of health and disease. Single cell proteomics has seen intense interest from mass spectrometrists, with a goal of quantifying the proteome of single cells by adapting current techniques used in bulk samples. To date, most method optimizations research has worked towards increasing the proteome coverage of single cells. One prominent technique multiplexes many individual cells into a single data acquisition event using isobaric labels. Accompanying the single cells, one label is typically used for a mixed set of many cells, called a carrier or boost channel. Although this improves peptide identification rates, several groups have examined the impact on quantitative accuracy as more cells are included in the carrier channel, e.g. 100x or 500x. This manuscript explores how impurities in the multiplexing reagent can lead to inaccurate quantification observed as a measurable signal in the wrong channel. We discover that the severe abundance differential between carrier and single cell, combined with the reagent impurities, can overshadow several channels typically used for single cells. For carrier amounts 100x and above, this contamination can be as abundant as true signal from a single cell. Therefore, we suggest limiting the carrier channel to a minimal amount and balance the goals of identification and quantification.

Patient characteristics, predictors and outcome of pacemaker patients upgraded to an implantable cardioverter defibrillator.

AIMS: Pacemaker (PM) patients may require a subsequent upgrade to an implantable cardioverter defibrillator (ICD). Limited data exists on this patient population. We sought to characterize this population, to assess predictors for ICD upgrade, and to report the outcome. METHODS: From our prospective PM and ICD implantation registry, all patients who underwent PM and/or ICD implantations at our center were analyzed. Patient characteristics and outcomes of PM patients with subsequent ICD upgrade were compared to age- and sex-matched patients with de novo ICD implantation, and to PM patients without subsequent upgrade. RESULTS: Of 1'301 ICD implantations, 60 (5%) were upgraded from PMs. Median time from PM implantation to ICD upgrade was 2.6 years (IQR 1.3-5.4). Of 2'195 PM patients, 28 patients underwent subsequent ICD upgrades, corresponding to an estimated annual incidence of an ICD upgrade of at least 0.33%. Lower LVEF (p = .05) and male sex (p = .038) were independent predictors for ICD upgrade. Survival without death, transplant and LVAD implantation were worse both for upgraded ICD patients compared to matched patients with de novo ICD implantation (p = .05), as well as for PM patients with subsequent upgrade compared to matched PM patients not requiring an upgrade (p = .036). CONCLUSIONS: One of 20 ICD implantations are upgrade of patients with a PM. At least one of 30 PM patients will require an ICD upgrade in the following 10 years. Predictors for ICD upgrade are male sex and lower LVEF at PM implantation. Upgraded patients have worse outcomes.

Expanding the Use of Continuous Sedation Until Death and Physician-Assisted Suicide.

The controversy over the equivalence of continuous sedation until death (CSD) and physician-assisted suicide/euthanasia (PAS/E) provides an opportunity to focus on a significant extended use of CSD. This extension, suggested by the equivalence of PAS/E and CSD, is designed to promote additional patient autonomy at the end-of-life. Samuel LiPuma, in his article, "Continuous Sedation Until Death as Physician-Assisted Suicide/Euthanasia: A Conceptual Analysis" claims equivalence between CSD and death; his paper is seminal in the equivalency debate. Critics contend that sedation follows proportionality protocols for which LiPuma's thesis does not adequately account. Furthermore, sedation may not eliminate consciousness, and as such LiPuma's contention that CSD is equivalent to neocortical death is suspect. We not only defend the equivalence thesis, but also expand it to include additional moral considerations. First, we explain the equivalence thesis. This is followed by a defense of the thesis against five criticisms. The third section critiques the current use of CSD. Finally, we offer two proposals that, if adopted, would broaden the use of PAS/E and CSD and thereby expand options at the end-of-life.

PCOS phenotype focus: phenotype D under the magnifying glass.

Polycystic ovary syndrome (PCOS) is defined as the combination of polycystic morphology, hyperandrogenism, and ovulatory disruption; this heterogeneity presents a conundrum for the medical community. The Rotterdam criteria have governed the diagnosis of PCOS, separating the patient cohort into four distinct phenotypes. It has been suggested that the lone normoandrogenic phenotype, so-called phenotype D, should not be classified as a PCOS subtype, with phenotypes A, B, and C displaying a hyperandrogenic biochemical and clinical profile thought to be characteristic of PCOS. To understand how to treat phenotype D patients, this review shines a spotlight on the phenotype, gathering various reports of how phenotype D is differentiated from the other PCOS phenotypes.

Trends Over Time in Recurrence Patterns and Survival Outcomes after Neoadjuvant Therapy and Surgery for Pancreatic Cancer.

OBJECTIVE: We aimed to determine if advances in neoadjuvant therapy affected recurrence patterns and survival outcomes after pancreatectomy for pancreatic ductal adenocarcinoma (PDAC). BACKGROUND: Data are limited on how modern multimodality therapy affects PDAC recurrence and post-recurrence survival. METHODS: Patients who received neoadjuvant therapy followed by curative-intent pancreatectomy for PDAC during 1998-2018 were identified. Treatments, recurrence sites and timing, and survival were compared between patients who completed neoadjuvant therapy and pancreatectomy in 1998-2004, 2005-2011, and 2012-2018. RESULTS: The study included 727 patients (203, 251, and 273 in the 1998-2004, 2005-2011, and 2012-2018 cohorts, respectively). Use of neoadjuvant induction chemotherapy increased over time, and regimens changed over time, with >80% of patients treated in 2012-2018 receiving FOLFIRINOX or gemcitabine with nab-paclitaxel. Overall, recurrence sites and incidence (67.5%, 66.1%, and 65.9%) remained stable, and 85% of recurrences occurred within 2 years of surgery. However, compared to earlier cohorts, the 2012-2018 cohort had lower conditional risk of recurrence in postoperative year 1 and higher risk in postoperative year 2. Overall survival increased over time (median, 30.6, 33.6, and 48.7 mo, P < 0.005), driven by improved post-recurrence overall survival (median, 7.8, 12.5, and 12.6 mo; 3-year rate, 7%, 10%, and 20%; P < 0.005). CONCLUSIONS: We observed changes in neoadjuvant therapy regimens over time and an associated shift in the conditional risk of recurrence from postoperative year 1 to postoperative year 2, although recurrence remained common. Overall survival and post-recurrence survival remarkably improved over time, reflecting improved multimodality regimens for recurrent disease.

Mitochondrial genomes of Pleistocene megafauna retrieved from recent sediment layers of two Siberian lakes.

Ancient environmental DNA (aeDNA) from lake sediments has yielded remarkable insights for the reconstruction of past ecosystems, including suggestions of late survival of extinct species. However, translocation and lateral inflow of DNA in sediments can potentially distort the stratigraphic signal of the DNA. Using three different approaches on two short lake sediment cores of the Yamal peninsula, West Siberia, with ages spanning only the past hundreds of years, we detect DNA and identified mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals-both species that have been extinct for thousands of years on the mainland. The occurrence of clearly identifiable aeDNA of extinct Pleistocene megafauna (e.g. >400 K reads in one core) throughout these two short subsurface cores, along with specificities of sedimentology and dating, confirm that processes acting on regional scales, such as extensive permafrost thawing, can influence the aeDNA record and should be accounted for in aeDNA paleoecology.

Human tear film protein sampling using soft contact lenses.

BACKGROUND: Human tear protein biomarkers are useful for detecting ocular and systemic diseases. Unfortunately, existing tear film sampling methods (Schirmer strip; SS and microcapillary tube; MCT) have significant drawbacks, such as pain, risk of injury, sampling difficulty, and proteomic disparities between methods. Here, we present an alternative tear protein sampling method using soft contact lenses (SCLs). RESULTS: We optimized the SCL protein sampling in vitro and performed in vivo studies in 6 subjects. Using Etafilcon A SCLs and 4M guanidine-HCl for protein removal, we sampled an average of 60 ± 31 µg of protein per eye. We also performed objective and subjective assessments of all sampling methods. Signs of irritation post-sampling were observed with SS but not with MCT and SCLs. Proteomic analysis by mass spectrometry (MS) revealed that all sampling methods resulted in the detection of abundant tear proteins. However, smaller subsets of unique and shared proteins were identified, particularly for SS and MCT. Additionally, there was no significant intrasubject variation between MCT and SCL sampling. CONCLUSIONS: These experiments demonstrate that SCLs are an accessible tear-sampling method with the potential to surpass current methods in sampling basal tears.