Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological ...targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.
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•Binding energies, association, and cleavage rates measured for >40,000 RISC targets•AGO2 tolerates large insertions in the target opposite the central region of the guide•Some guide:target mismatches enhance the single-turnover RISC cleavage rate•In vitro measured biochemical parameters explain knockdown in cells
By high-throughput, quantitative characterization of binding and cleavage for >40,000 distinct RISC targets, Becker et al. reveal principles of miRNA regulation and siRNA function. These data enable construction of quantitative models of binding and cleavage and are used to explain mRNA knockdown in cells.
The foremost document that comprehensively reports on biological control introductions against weeds—‘Biological control of weeds: a world catalogue of agents and their target weeds’—has been updated ...and now includes all deliberate releases made through 2012. It includes data on 1555 intentional releases of 468 biological control agent species used against 175 species of target weeds in 48 plant families, in 90 countries. For 55 (31.4%) of the target weed species, only one biocontrol agent was introduced. The largest number of agent species (44) was introduced for the biological control of
Lantana camara
(Verbenaceae). Three insect orders (Coleoptera, Lepidoptera and Diptera) comprised about 80% of all biocontrol agent species released and releases made. Of the 468 biocontrol agent species introduced, 332 (70.9%) established in at least one instance. Of the 313 species, for which impact could be categorized, 172 (55.0%) caused medium, variable or heavy levels of damage (impacts). Of all releases made through 2012, 982 (63.2%) led to establishment. Forty-two releases were judged too early post-release to categorize impact, leaving 940 releases for which impact analyses were conducted. Similar to agent species, approximately half of the established releases (503 or 53.5%) caused medium, variable or heavy levels of damage on the target weeds, and almost a quarter of releases (225 or 23.9%) caused heavy impact. Across all countries and regions, 65.7% of the weeds targeted for biological control experienced some level of control. These data indicate the value of this practice, on its own, or as a supplement to other methods, in the management of invasive plants.
The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall ...health
. The intesting has a length of over nine metres, along which there are differences in structure and function
. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.
Abstract Purpose We sought to project the number of primary care physicians required to meet US health care utilization needs through 2025 after passage of the Affordable Care Act. Methods In this ...projection of workforce needs, we used the Medical Expenditure Panel Survey to calculate the use of office-based primary care in 2008. We used US Census Bureau projections to account for demographic changes and the American Medical Association's Masterfile to calculate the number of primary care physicians and determine the number of visits per physician. The main outcomes were the projected number of primary care visits through 2025 and the number of primary care physicians needed to conduct those visits. Results Driven by population growth and aging, the total number of office visits to primary care physicians is projected to increase from 462 million in 2008 to 565 million in 2025. After incorporating insurance expansion, the United States will require nearly 52,000 additional primary care physicians by 2025. Population growth will be the largest driver, accounting for 33,000 additional physicians, while 10,000 additional physicians will be needed to accommodate population aging. Insurance expansion will require more than 8,000 additional physicians, a 3% increase in the current workforce. Conclusions Population growth will be the greatest driver of expected increases in primary care utilization. Aging and insurance expansion will also contribute to utilization, but to a smaller extent.
Diet has important effects on normal physiology and the potential deleterious effects of high fat diets and obesity on male reproductive health are being increasingly described. We conducted a ...histological review of the effects of chronic high fat (HF) diet (using a mouse model fed a 45% fat diet for 21 weeks) with a discovery proteomic study to assess for changes in the abundance of proteins in the testis. Mice on a HF diet became obese and developed glucose intolerance. Using mass spectrometry, we identify 102 proteins affected in the testis of obese mice. These included structural proteins important for the blood testis barrier (filamin A, FLNA), proteins involved in oxidative stress responses (spermatogenesis associated 20, SPATA-20) and lipid homoeostasis (sterol regulatory element-binding protein 2, SREBP2 and apolipoprotein A1, APOA1). In addition, an important regulator protein paraspeckle component 1, PSPC-1, which interacts with the androgen receptor was significantly downregulated. Proteomic data was validated using both Western blotting and immunostaining which confirmed and localised protein expression in both mouse and human testis using biopsy specimens. This study focused mainly on the abnormalities that occurred at the protein level and as a result, we have identified several candidate proteins and conducted pathway analysis around the effects of HF diet on the testis providing novel insights not previously described. Some of the identified targets could be targeted therapeutically and future work is directed in this area.
To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated single-cell chromatin ...accessibility profiles and single-cell transcriptomes from 1,000 to 10,000 cells per sample for 48 polyps, 27 normal tissues and 6 CRCs collected from patients with or without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from homeostasis to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T cells and a subtype of pre-cancer-associated fibroblasts. In the cancerous state, we observe T cell exhaustion, RUNX1-regulated cancer-associated fibroblasts and increasing accessibility associated with HNF4A motifs in epithelia. DNA methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.
High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the ...landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.
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•A thermodynamic model quantitatively predicts PUM1/2 binding to any RNA sequence•Factors beyond simple recognition of consecutive residues influence binding•Comparison to X-linking data reveals thermodynamic control of PUM2 binding in cells•Analysis of RNA structure effects suggests disruption of RNA structure in cells
As RNA-binding proteins play key roles in cellular regulation, quantitative approaches are needed to capture RNA-protein interaction landscapes. Jarmoskaite et al. establish a quantitative model that predicts human PUM1/2 protein-RNA affinities and cellular PUM2-RNA occupancies, suggesting a continuous binding landscape negligibly affected by RNA structure and kinetic factors.
ABSTRACT PURPOSE The purpose of this study was to calculate the projected primary care physician shortage, determine the amount and composition of residency growth needed, and estimate the impact of ...retirement age and panel size changes. METHODS We used the 2010 National Ambulatory Medical Care Survey to calculate utilization of ambulatory primary care services and the US Census Bureau to project demographic changes. To determine the baseline number of primary care physicians and the number retiring at 66 years, we used the 2014 American Medical Association Masterfile. Using specialty board and American Osteopathic Association figures, we estimated the annual production of primary care residents. To calculate shortages, we subtracted the accumulated primary care physician production from the accumulated number of primary care physicians needed for each year from 2015 to 2035. RESULTS More than 44,000 primary care physicians will be needed by 2035. Current primary care production rates will be unable to meet demand, resulting in a shortage in excess of 33,000 primary care physicians. Given current production, an additional 1,700 primary care residency slots will be necessary by 2035. A 10% reduction in the ratio of population per primary care physician would require more than 3,000 additional slots by 2035, whereas changing the expected retirement age from 66 years to 64 years would require more than 2,400 additional slots. CONCLUSIONS To eliminate projected shortages in 2035, primary care residency production must increase by 21% compared with current production. Delivery models that shift toward smaller ratios of population to primary care physicians may substantially increase the shortage.
Abstract Purpose Loneliness has important health consequences. Little is known, however, about loneliness in primary care patient populations. This study describes the prevalence of loneliness in ...patients presenting for primary care and associations with self-reported demographic factors, health care utilization, and health-related quality of life. Methods We conducted cross-sectional surveys of adults presenting for routine care to outpatient primary care practices in 2 diverse practice-based research networks. The 3-item University of California, Los Angeles Loneliness Scale was utilized to determine loneliness. Results The prevalence of loneliness was 20% (246/1,235). Loneliness prevalence was inversely associated with age ( P <.01) and less likely in those who were married ( P <.01) or employed ( P <.01). Loneliness was more common in those with lower health status ( P <.01), including when adjusting for employment and relationship status (odds ratio OR = 1.05; 95% CI, 1.03–1.07). Primary care visits (OR = 1.07; 95% CI, 1.03–1.10), urgent care/emergency department visits (OR = 1.24; 95% CI, 1.12–1.38), and hospitalizations (OR = 1.15; 95% CI, 1.01–1.31) were associated with loneliness status. There was no significant difference in rates of loneliness between sexes ( P = .08), racial categories ( P = .57), or rural and urban respondents ( P = .42). Conclusions Our findings demonstrate that loneliness is common in primary care patients and is associated with adverse health consequences including poorer health status and greater health care utilization. Further work is needed to understand the value of screening for and using interventions to treat loneliness in primary care.
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