Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics

New therapeutic strategies are needed to treat infections caused by drug-resistant bacteria, which constitute a major growing threat to human health. Here, we use a high-throughput technology to identify combinatorial genetic perturbations that can enhance the killing of drug-resistant bacteria with antibiotic treatment. This strategy, Combinatorial Genetics En Masse (CombiGEM), enables the rapid generation of high-order barcoded combinations of genetic elements for high-throughput multiplexed characterization based on next-generation sequencing. We created ~34,000 pairwise combinations of Escherichia coli transcription factor (TF) overexpression constructs. Using Illumina sequencing, we identified diverse perturbations in antibiotic-resistance phenotypes against carbapenem-resistant Enterobacteriaceae. Specifically, we found multiple TF combinations that potentiated antibiotic killing by up to 10[superscript 6]-fold and delivered these combinations via phagemids to increase the killing of highly drug-resistant E. coli harboring New Delhi metallo-beta-lactamase-1. Moreover, we constructed libraries of three-wise combinations of transcription factors with >4 million unique members and demonstrated that these could be tracked via next-generation sequencing. We envision that CombiGEM could be extended to other model organisms, disease models, and phenotypes, where it could accelerate massively parallel combinatorial genetics studies for a broad range of biomedical and biotechnology applications, including the treatment of antibiotic-resistant infections.National Institutes of Health (U.S.) (New Innovator Award DP2 OD008435)United States. Office of Naval ResearchEllison Medical Foundation (New Scholar in Aging Award)Henry L. and Grace Doherty Charitable Foundatio

Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae

Position of siRNAs along the Vta2 gene of V. dahliae. The position of different siRNAs designed to target this gene is shown in this figure. Sequence underlined with different colors shows different siRNAs. (JPG 7797 kb

Identification and Functional Analysis of Two New Mutant BnFAD2 Alleles That Confer Elevated Oleic Acid Content in Rapeseed

Rapeseed (Brassica napus L.) is a vital oil crop worldwide. High oleic acid content is a desirable quality trait for rapeseed oil, which makes it more beneficial to human health. However, many germplasm resources with high oleic acid content in rapeseed have not been evaluated with regard to their genotypes, making it difficult to select the best strains with this trait for the breeding of high oleic acid rapeseed variety. This work was to explore the gene-regulation mechanism of this trait using a new super-high oleic acid content (∼85%) line N1379T as genetic material. In this study, the sequences of four homologous fatty acid desaturase (BnFAD2) genes were compared between super-high (∼85%, N1379T) and normal (∼63%) oleic acid content lines. Results showed that there were two single-nucleotide polymorphisms (SNPs) in BnFAD2-1 and BnFAD2-2, respectively, which led to the amino acid changes (E106K and G303E) in the corresponding proteins. Functional analysis of both genes in yeast confirmed that these SNPs were loss-of-function mutations, thus limiting the conversion of oleic acid to linoleic acid and resulting in the considerable accumulation of oleic acid. Moreover, two specific cleaved amplified polymorphic sequences (CAPS) markers for the two SNPs were developed to identify genotypes of each line in the F2 and BC1 populations. Furthermore, these two mutant loci of BnFAD2-1 and BnFAD2-2 genes were positively associated with elevated oleic acid levels and had a similar effect with regard to the increase of oleic acid content. Taken together, these two novel SNPs in two different BnFAD2 genes jointly regulated the high oleic acid trait in this special germplasm. The study provided insight into the genetic regulation involved in oleic acid accumulation and highlighted the use of new alleles of BnFAD2-1 and BnFAD2-2 in breeding high oleic acid rapeseed varieties

Probing Quantum Confinement of Single-Walled Carbon Nanotubes by Resonant Soft-X-Ray Emission Spectroscopy

[[abstract]]We report the band-structure changes near Fermi level for single-walled carbon nanotubes (SWNTs) with diameters down to 1 nm from the study of soft-x-ray absorption and resonant emission spectroscopy. The observed quantum confinement of SWNTs affects both pi and sigma bands and bandgap through the rehybridization of pi and sigma orbitals. The significant changes of electronic structure are proved to be a measure for the mean diameter of the macroscopic amounts of SWNTs. (C) 2008 American Institute of Physics.[[notice]]補正完畢[[booktype]]紙

Onset of Tethered Chain Overcrowding

We proposed an approach to precisely control the density of tethered chains on solid substrates using PEO-b-PS and PLLA-b-PS. As the crystallization temperature T-x increased, the PEO or PLLA lamellar crystal thickness d(L) increased as well as the reduced tethering density (σ) over tilde of the PS chains. The onset of tethered PS chains overcrowding in solution occurs at (σ) over tilde*similar to3.7-3.8 as evidenced by an abrupt change in the slope between (d(L))(-1) and T-x. This results from the extra surface free energy created by the tethered chain that starts to affect the growth barrier of the crystalline blocks

Determination of reference intervals of serum levels of human epididymis protein 4 (HE4) in Chinese women

The detailed numbers of subjects from different centers. (DOCX 17.3 kb

Onset of tethered chain overcrowding

Physical Review Letters, 93(2): pp. 028301-1 -- 028301-4. Retrieved September 19, 2006 from http://www.materials.drexel.edu/SRG/chrisli.html. DOI: http://dx.doi.org/10.1103/PhysRevLett.93.028301We proposed an approach to precisely control the density of tethered chains on solid substrates using PEO-b-PS and PLLA-b-PS. As the crystallization temperature T-x increased, the PEO or PLLA lamellar crystal thickness d(L) increased as well as the reduced tethering density (σ) over tilde of the PS chains. The onset of tethered PS chains overcrowding in solution occurs at (σ) over tilde*similar to3.7-3.8 as evidenced by an abrupt change in the slope between (d(L))(-1) and T-x. This results from the extra surface free energy created by the tethered chain that starts to affect the growth barrier of the crystalline blocks

Clinical Characteristics of 26 Human Cases of Highly Pathogenic Avian Influenza A (H5N1) Virus Infection in China

BACKGROUND: While human cases of highly pathogenic avian influenza A (H5N1) virus infection continue to increase globally, available clinical data on H5N1 cases are limited. We conducted a retrospective study of 26 confirmed human H5N1 cases identified through surveillance in China from October 2005 through April 2008. METHODOLOGY/PRINCIPAL FINDINGS: Data were collected from hospital medical records of H5N1 cases and analyzed. The median age was 29 years (range 6-62) and 58% were female. Many H5N1 cases reported fever (92%) and cough (58%) at illness onset, and had lower respiratory findings of tachypnea and dyspnea at admission. All cases progressed rapidly to bilateral pneumonia. Clinical complications included acute respiratory distress syndrome (ARDS, 81%), cardiac failure (50%), elevated aminotransaminases (43%), and renal dysfunction (17%). Fatal cases had a lower median nadir platelet count (64.5 x 10(9) cells/L vs 93.0 x 10(9) cells/L, p = 0.02), higher median peak lactic dehydrogenase (LDH) level (1982.5 U/L vs 1230.0 U/L, p = 0.001), higher percentage of ARDS (94% [n = 16] vs 56% [n = 5], p = 0.034) and more frequent cardiac failure (71% [n = 12] vs 11% [n = 1], p = 0.011) than nonfatal cases. A higher proportion of patients who received antiviral drugs survived compared to untreated (67% [8/12] vs 7% [1/14], p = 0.003). CONCLUSIONS/SIGNIFICANCE: The clinical course of Chinese H5N1 cases is characterized by fever and cough initially, with rapid progression to lower respiratory disease. Decreased platelet count, elevated LDH level, ARDS and cardiac failure were associated with fatal outcomes. Clinical management of H5N1 cases should be standardized in China to include early antiviral treatment for suspected H5N1 cases

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing