The development of multifunctional nanoparticles that have dual capabilities of tumor imaging and delivering therapeutic agents into tumor cells holds great promises for novel approaches for tumor imaging and therapy. We have engineered urokinase plasminogen activator receptor (uPAR) targeted biodegradable nanoparticles using a size uniform and amphiphilic polymer-coated magnetic iron oxide (IO) nanoparticle conjugated with the amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA), which is a high affinity natural ligand for uPAR. We further developed methods to encapsulate hydrophobic chemotherapeutic drugs into the polymer layer on the IO nanoparticles, making these targeted magnetic resonance imaging (MRI) sensitive nanoparticles drug delivery vehicles. Using a fluorescent drug doxorubicin (Dox) as a model system, we showed that this hydrophobic drug can be efficiently encapsulated into the uPAR-targeted IO nanoparticles. This class of Dox-loaded nanoparticles has a compact size and is stable in pH 7.4 buffer. However, encapsulated Doxcan be released from the nanoparticles at pH 4.0 to 5.0 within 2 hrs. In comparison with the effect of equivalent dosage of free drug or non-targeted IO-Dox nanoparticles, uPAR-targeted IO-Dox nanoparticles deliver higher levels of Dox into breast cancer cells and produce a stronger inhibitory effect on tumor cell growth. Importantly, Dox-loaded IO nanoparticles maintain their T2 MRI contrast effect after being internalized into the tumor cells due to their significant susceptibility effect in the cells, indicating that this drug delivery nanoparticle has the potential to be used as targeted therapeutic imaging probes for monitoring the drug delivery using MRI.

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Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of lameness in commercial broiler chickens worldwide. BCO represents substantial production loss and welfare issues of chickens. The bacterial species or communities underlying BCO pathogenesis still remain to be fully characterized. To gain insights on blood microbiota in broilers and its potential association with BCO, blood samples collected from healthy (n = 240) and lame (n = 12) chickens were analyzed by deep sequencing of 16S RNA genes. The chicken blood microbiota were dominated by Proteobacteria (60.58% ± 0.65) followed by Bactroidetes (13.99% ± 0.29), Firmicutes (11.45% ± 0.51), Actinobacteria (10.21% ± 0.37) and Cyanobacteria (1.96% ± 0.21) that constituted 98.18% (± 0.22) of the whole phyla. The bacterial communities consist of 30–40 OTUs in the blood of broiler chickens, regardless of ages and other environmental or host conditions, and the blood microbiomes of BCO chickens were largely distinct from those of healthy chickens. In addition, Linear discriminant analysis (LDA) effect size (LEfSe) method revealed that Staphylococcus, Granulicatella, and Microbacterium were significantly enriched in BCO chickens as compared to healthy chickens. The results from this study have significant implications in understanding blood microbiota present in broiler chickens and its potential role in BCO pathogenesis.

ABSTRACTAs more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturatingSalmonella entericaserotype Typhimurium mutant library recovered from selection under 3 differentin vitrogrowth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes inS. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD,glnL,recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes inSalmonellaand the other approximately 70 bacterial species for which EZ:Tn5mutagenesis has been established.

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A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.

Background: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.

Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers.