Analyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in molecular profiling of ...cellular constituents, spatially mapping them remains a disjointed and specialized machinery-intensive process, relying on either light microscopy or direct physical registration. Here, we demonstrate DNA microscopy, a distinct imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences and infers physical images of the original transcripts at cellular resolution with precise sequence information. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.
Display omitted
•DNA microscopy is a distinct imaging modality that encodes physical images into DNA•A stand-alone reaction first writes inter-molecular proximities into DNA•An algorithm then reconstructs a supra-molecular image from this DNA recording•DNA microscopy thereby constitutes a chemically encoded microscopy system
DNA microscopy is an optics-free imaging method based on chemical reactions and a computational algorithm to infer spatial organization of transcripts while simultaneously preserving full sequence information.
According to quantum mechanics, a harmonic oscillator can never be completely at rest. Even in the ground state, its position will always have fluctuations, called the zero-point motion. Although the ...zero-point fluctuations are unavoidable, they can be manipulated. Using microwave frequency radiation pressure, we have manipulated the thermal fluctuations of a micrometer-scale mechanical resonator to produce a stationary quadrature-squeezed state with a minimum variance of 0.80 times that of the ground state. We also performed phase-sensitive, back-action evading measurements of a thermal state squeezed to 1.09 times the zero-point level. Our results are relevant to the quantum engineering of states of matter at large length scales, the study of decoherence of large quantum systems, and for the realization of ultrasensitive sensing of force and motion.
Background. Recent studies have suggested that community-associated methicillin-resistant Staphylococcus aureus (MRSA) infection is encroaching on health care settings. We describe the epidemiology ...of hospital-onset community-associated MRSA bloodstream infections using phenotypic and genotypic analysis. Methods. Using an update of an established rule derived from antibiotic susceptibilities, we inferred genotypes (i.e., community CG or hospital HG) for 208 MRSA isolates from hospital-onset (>72 h after hospital admission) bloodstream infections during 2000–2006. We compared demographic characteristics, risk factors, and outcomes of patients infected with CG or HG strains. Results. Total hospital-onset MRSA bloodstream infection incidence density rates for the periods January 2000–June 2003 and July 2003–December 2006 (0.215 cases per 1000 patient-days and 0.207 cases per 1000 patient-days, respectively) were stable (risk ratio, 1.0; 95% confidence interval, 0.7–1.3; P=.79, period 2 vs. period 1). However, the risk that these bloodstream infections were due to CG strains doubled (risk ratio, 1.9; 95% confidence interval, 1.2–3.1; P=.01), whereas the risk due to HG strains decreased (risk ratio, 0.7; 95% confidence interval, 0.46–0.93; P=.02). After adjustment for comorbidities in multivariate analysis, no significant risk factors for or outcomes of infections due to CG versus HG strains were detected. Patients infected with HG strains showed a trend toward later day of acquisition of a positive blood culture, and those infected with CG strains showed trend toward greater risk of intensive care unit admission. Conclusion. Although total hospital-onset MRSA bloodstream infection rates were relatively stable during 2000–2006, CG strains were responsible for an increasing proportion of cases (from 24% to 49%), suggesting replacement of traditional hospital-associated strains. For most risk factors and outcomes, patients infected with CG and HG strains were similar, suggesting that, thus far, CG strains are behaving like their traditional hospital-associated counterparts.
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting ...specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
Background. Organisms resistant to antimicrobials continue to emerge and spread. This study was performed to measure the medical and societal cost attributable to antimicrobial-resistant infection ...(ARI). Methods. A sample of high-risk hospitalized adult patients was selected. Measurements included ARI, total cost, duration of stay, comorbidities, acute pathophysiology, Acute Physiology and Chronic Health Evaluation III score, intensive care unit stay, surgery, health care-acquired infection, and mortality. Hospital services used and outcomes were abstracted from electronic and written medical records. Medical costs were measured from the hospital perspective. A sensitivity analysis including 3 study designs was conducted. Regression was used to adjust for potential confounding in the random sample and in the sample expanded with additional patients with ARI. Propensity scores were used to select matched control subjects for each patient with ARI for a comparison of mean cost for patients with and without ARI. Results. In a sample of 1391 patients, 188 (13.5%) had ARI. The medical costs attributable to ARI ranged from $18,588 to $29,069 per patient in the sensitivity analysis. Excess duration of hospital stay was 6.4–12.7 days, and attributable mortality was 6.5%. The societal costs were $10.7–$15.0 million. Using the lowest estimates from the sensitivity analysis resulted in a total cost of $13.35 million in 2008 dollars in this patient cohort. Conclusions. The attributable medical and societal costs of ARI are considerable. Data from this analysis could form the basis for a more comprehensive evaluation of the cost of resistance and the potential economic benefits of prevention programs.
In this cluster-randomized study at ICUs in six hospitals, chlorhexidine-impregnated washcloths were associated with significantly lower rates of bloodstream infections and acquisition of ...multidrug-resistant organisms than were nonantimicrobial washcloths.
Multidrug-resistant organisms (MDROs), including methicillin-resistant
Staphylococcus aureus
(MRSA) and vancomycin-resistant enterococcus (VRE), have become endemic in many acute care and long-term care facilities.
1
–
5
Infections with these organisms are often difficult to treat, owing to a dwindling armamentarium of active antimicrobial agents. The Centers for Disease Control and Prevention (CDC) has promulgated a variety of strategies, including hand hygiene and the use of isolation precautions, to limit the spread of these organisms among patients, but these strategies require consistent adherence to practices by large numbers of health care personnel during frequent patient encounters and can be difficult to sustain.
6
In . . .
Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either ...newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level—which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalent-inactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.
Background. Klebsiella pneumoniae carbapenemase (KPC)—producing Enterobacteriaceae are an emerging antibiotic resistance threat with demonstrated epidemic potential. Methods. We conducted an outbreak ...investigation of KPC-producing Enterobacteriaceae among patients of acute and long-term acute care hospitals (LTACHs) in 4 adjacent counties in Indiana and Illinois from 1 January 2008 through 31 December 2008 (cases). The study used traditional and molecular epidemiologic methods and an adaptation of social network analysis ("exposure network analysis"). Results. Clinical records for 40 (95%) of 42 patients were available. Patients were mostly older with multiple comorbid conditions. Eleven patients (27.5%) died during the index hospitalization or were discharged to hospice; 23 (57.5%) were discharged to a nursing home, and 4 (10.0%) were discharged to home. One LTACH (LTACH-A) was central to the regional outbreak: 24 (60%) of 40 cases were linked to LTACH-A, and at least 10 patients (25%) acquired KPC there. Of 16 cases not linked to LTACH-A, 12 (75%) were linked to 3 nursing homes. Only 4 patients (10%) definitely acquired KPC during an acute care hospital stay. Molecular typing revealed the 31 available KPC-positive K. pneumoniae isolates to be similar and to cluster with epidemic multilocus sequence type 258; 2 KPC-positive Escherichia coli isolates were unique. Conclusions. We observed extensive transfer of KPC-positive patients throughout the exposure network of 14 acute care hospitals, 2 LTACHs, and 10 nursing homes. Although few cases were identified at most institutions, many facilities were affected. Successful control of KPC-producing Enterobacteriaceae will require a coordinated, regional effort among acute and long-term health care facilities and public health departments.
PDF
