Bacillus anthracis genome organization in light of whole transcriptome sequencing

Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity

Temperature mapping above and below air film-cooled thermal barrier coatings using phosphor thermometry

Thermal barrier coatings (TBCs) are typically used in conjunction with air film cooling to maximize overall cooling effectiveness and reliability while minimizing sacrifices in engine performance. The effects of thermal barrier coating (TBC) thermal protection and air film cooling effectiveness have usually been studied separately; however, their contributions to combined cooling effectiveness are interdependent and are not simply additive. The combined cooling effectiveness is always less than the sum of the cooling effectiveness of stand-alone TBC protection and stand-alone air film cooling. These diminishing returns arise because adding the thermally insulating TBC between the cooling air and the surface to be cooled reduces the air film cooling effectiveness and because the air film cooling reduces the heat flux through the TBC and therefore reduces the temperature difference sustained across the TBC thickness. Due to these considerations, combined cooling effectiveness must be measured to achieve an optimum balance between TBC thermal protection and air film cooling. In this investigation, temperature mapping above and below air film-cooled TBCs was performed using luminescence lifetime imaging-based phosphor thermometry. Measurements were performed in the NASA GRC Mach 0.3 burner rig on a TBC-coated plate using a scaled-up cooling hole geometry where both the hot mainstream gas temperature and the blowing ratio were varied. Surface temperature maps were obtained from a Cr-doped GdAlO3 thermographic phosphor deposited on the surface of the electron-beam vapor-deposited yttria-stabilized zirconia (YSZ) TBC. From separate plates, temperature maps from the bottom of the TBC were obtained from a thin Er-doped YSZ layer integrated into the TBC below the overlying undoped YSZ. Procedures for temperature and cooling effectiveness mapping above and below the air film-cooled TBC surface are described. Most importantly, these measurements enable mapping the combined cooling effectiveness below the TBC, which is more important than surface cooling effectiveness when there is a barrier coating between the hot mainstream gas and the surface that needs thermal protection. Advantages of the luminescence lifetime imaging method over infrared thermography, as well as its limitations to steady-state conditions are discussed

Novel clone selection technique reveals heterogeneity among HEK293T cells engineered to produce therapeutic extracellular vesicles

HEK293T cells have been engineered to produce extracellular vesicles (EVs) that deliver miR-199a-3p to CD44+ hepatocellular carcinoma cells. Restoration of this miRNA has been shown to slow cancer progression in-vitro. Isolation and analysis of EVs from cell culture media containing selection agent revealed that the number of miRNA-199a-3p copies was less than the number of cells in culture suggesting that not all cells produce therapeutic EVs. Therefore, therapeutic EV production can be significantly increased by selecting the HEK293T clones that produce the most therapeutic EVs. While clone selection is traditionally accomplished by cell analysis techniques such as fluorescence activated cell sorting (FACS), detection of therapeutic EVs poses a unique challenge in that cellular expression of miRNA-199a-3p does not necessarily correlate to the amount of exosomal miRNA-199a-3p. In response to this challenge, a fibrous microwell array was developed to screen thousands of clones for therapeutic EV productivity (figure 1). The fibrous microwell system is able to evaluate cell growth rate under fluid shear stress, EV productivity and EV characterization using fluorescently labeled antibodies or cationic lipoplex nanoparticles (detect presence of miRNA-199a-3p inside captured EVs produced by single clones). The most productive clones can be released from the microwells and grown in large scale cell culture to significantly increase therapeutic EV production. Please click Additional Files below to see the full abstract

Strain prioritization and genome mining for enediyne natural products

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family

Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice

RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments

LabelBench: A Comprehensive Framework for Benchmarking Adaptive Label-Efficient Learning

Labeled data are critical to modern machine learning applications, but obtaining labels can be expensive. To mitigate this cost, machine learning methods, such as transfer learning, semi-supervised learning and active learning, aim to be label-efficient: achieving high predictive performance from relatively few labeled examples. While obtaining the best label-efficiency in practice often requires combinations of these techniques, existing benchmark and evaluation frameworks do not capture a concerted combination of all such techniques. This paper addresses this deficiency by introducing LabelBench, a new computationally-efficient framework for joint evaluation of multiple label-efficient learning techniques. As an application of LabelBench, we introduce a novel benchmark of state-of-the-art active learning methods in combination with semi-supervised learning for fine-tuning pretrained vision transformers. Our benchmark demonstrates better label-efficiencies than previously reported in active learning. LabelBench's modular codebase is open-sourced for the broader community to contribute label-efficient learning methods and benchmarks. The repository can be found at: https://github.com/EfficientTraining/LabelBench

Evaluation of noise regression techniques in resting-state fMRI studies using data of 434 older adults

Subject motion is a well-known confound in resting-state functional MRI (rs-fMRI) and the analysis of functional connectivity. Consequently, several clean-up strategies have been established to minimize the impact of subject motion. Physiological signals in response to cardiac activity and respiration are also known to alter the apparent rs-fMRI connectivity. Comprehensive comparisons of common noise regression techniques showed that the Independent Component Analysis based strategy for Automatic Removal of Motion Artifacts (ICA-AROMA) was a preferred pre-processing technique for teenagers and adults. However, motion and physiological noise characteristics may differ substantially for older adults. Here, we present a comprehensive comparison of noise-regression techniques for older adults from a large multi-site clinical trial of exercise and intensive pharmacological vascular risk factor reduction. The Risk Reduction for Alzheimer\u27s Disease (rrAD) trial included hypertensive older adults (60-84 years old) at elevated risk of developing Alzheimer\u27s Disease (AD). We compared the performance of censoring, censoring combined with global signal regression, non-aggressive and aggressive ICA-AROMA, as well as the Spatially Organized Component Klassifikator (SOCK) on the rs-fMRI baseline scans from 434 rrAD subjects. All techniques were rated based on network reproducibility, network identifiability, edge activity, spatial smoothness, and loss of temporal degrees of freedom (tDOF). We found that non-aggressive ICA-AROMA did not perform as well as the other four techniques, which performed table with marginal differences, demonstrating the validity of these techniques. Considering reproducibility as the most important factor for longitudinal studies, given low false-positive rates and a better preserved, more cohesive temporal structure, currently aggressive ICA-AROMA is likely the most suitable noise regression technique for rs-fMRI studies of older adults

Study on the Self-Repairing Effect of Nanoclay in Powder Coatings for Corrosion Protection

Powder coatings are a promising, solvent-free alternative to traditional liquid coatings due to the superior corrosion protection they provide. This study investigates the effects of incorporating montmorillonite-based nanoclay additives with different particle sizes into polyester/triglycidyl isocyanurate (polyester/TGIC) powder coatings. The objective is to enhance the corrosion-protective function of the coatings while addressing the limitations of commonly employed epoxy-based coating systems that exhibit inferior UV resistance. The anti-corrosive and surface qualities of the coatings were evaluated via neutral salt spray tests, electrochemical measurements, and surface analytical techniques. Results show that the nanoclay with a larger particle size of 18.38 µm (D50, V) exhibits a better barrier effect at a lower dosage of 4%, while a high dosage leads to severe defects in the coating film. Interestingly, the coating capacitance is found, via electrochemical impedance spectroscopy, to decrease during the immersion test, indicating a self-repairing capability of the nanoclay, arising from its swelling and expansion. Neutral salt spray tests suggest an optimal nanoclay dosage of 2%, with the smaller particle size (8.64 µm, D50, V) nanoclay providing protection for 1.5 times as many salt spray hours as the nanoclay with a larger particle size. Overall, incorporating montmorillonite-based nanoclay additives is suggested to be a cost-effective approach for significantly enhancing the anti-corrosive function of powder coatings, expanding their application to outdoor environments