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

The Impact of PISA on Students' Learning: a Chinese Perspective

This thesis investigates PISA’s impact on students’ learning in a local context, Fangshan District of Beijing, in China. PISA’s growing influence on educational policymaking in domestic education systems has been widely discussed, but concerns about policy borrowing and PISA’s governing power on education have been raised. However, these discussions rarely look further into local contexts to investigate PISA’s impact on students’ learning. Through mixed methods research, this thesis presents an investigation into the impact PISA has had on students’ learning and how this impact occurs. A conceptual framework employing theories about washback effect and ecological systems theory was developed to underpin the research. Sixteen local educational policymakers and practitioners were interviewed to identify how PISA is used in the local context, and to gather their views on the perceived impact of PISA on students’ learning. Fangshan PISA data were used to triangulate their perceptions, and also to expand the understanding of their use and translation of PISA data in policymaking. Thematic analysis of interview data reveals that PISA is perceived as a new perspective, which is different from domestic assessment in some aspects, for benchmarking the quality of local education. PISA is also used as an impetus motivating local initiatives to improve educational quality, in which some PISA concepts are integrated. The mechanism of PISA’s impact on students’ learning was conceptualised via making the factors of different contextual layers which negotiate PISA’s impact explicit. Interviewees perceived that through school enactment of the local PISA-motivated initiatives, and reforms of national curriculum and assessment, to some extent, students’ learning has been gradually fostered. These perceptions are largely supported by the trend analysis and multilevel modelling of PISA data. Research findings also indicate the challenges that the local PISA data users face for appropriately interpreting and translating PISA data to inform educational policymaking

Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus

The marine cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism in the oligotrophic oceans, and a model system in marine microbial ecology. Here we report 27 new whole genome sequences (2 complete and closed; 25 of draft quality) of cultured isolates, representing five major phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from diverse regions of the oceans, facilitating studies of the drivers of microbial diversity—both in the lab and in the field. To improve the utility of these genomes for comparative genomics, we also define pre-computed clusters of orthologous groups of proteins (COGs), indicating how genes are distributed among these and other publicly available Prochlorococcus genomes. These data represent a significant expansion of Prochlorococcus reference genomes that are useful for numerous applications in microbial ecology, evolution and oceanography.Gordon and Betty Moore Foundation (Grant GBMR #495.01)National Science Foundation (U.S.) (Grant OCE-1153588)National Science Foundation (U.S.) (Grant OCE-0425602)National Science Foundation (U.S.) (Grant DBI-0424599)Center for Microbial Oceanography: Research and Educatio

Hierarchical Fashion Design with Multi-stage Diffusion Models

Cross-modal fashion synthesis and editing offer intelligent support to fashion designers by enabling the automatic generation and local modification of design drafts.While current diffusion models demonstrate commendable stability and controllability in image synthesis,they still face significant challenges in generating fashion design from abstract design elements and fine-grained editing.Abstract sensory expressions, \eg office, business, and party, form the high-level design concepts, while measurable aspects like sleeve length, collar type, and pant length are considered the low-level attributes of clothing.Controlling and editing fashion images using lengthy text descriptions poses a difficulty.In this paper, we propose HieraFashDiff,a novel fashion design method using the shared multi-stage diffusion model encompassing high-level design concepts and low-level clothing attributes in a hierarchical structure.Specifically, we categorized the input text into different levels and fed them in different time step to the diffusion model according to the criteria of professional clothing designers.HieraFashDiff allows designers to add low-level attributes after high-level prompts for interactive editing incrementally.In addition, we design a differentiable loss function in the sampling process with a mask to keep non-edit areas.Comprehensive experiments performed on our newly conducted Hierarchical fashion dataset,demonstrate that our proposed method outperforms other state-of-the-art competitors

Identification of Saccharomyces cerevisiae Spindle Pole Body Remodeling Factors

The Saccharomyces cerevisiae centrosome or spindle pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes

Braveheart, a Long Noncoding RNA Required for Cardiovascular Lineage Commitment

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.Damon Runyon Cancer Research Foundation (DRG 2032-09)Damon Runyon Cancer Research Foundation (DFS 04-12)European Molecular Biology Organization (Long-term Fellowship)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098179)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098188)Smith Family FoundationPew Charitable Trusts. Program in the Biomedical Science

Massive and destructive T cell response to homeostatic cue in CD24-deficient lymphopenic hosts

In response to a lymphopenic cue, T lymphocytes undergo a slow-paced homeostatic proliferation in an attempt to restore T cell cellularity. The molecular interaction that maintains the pace of homeostatic proliferation is unknown. In this study, we report that in lymphopenic CD24-deficient mice, T cells launch a massive proliferation that results in the rapid death of the recipient mice. The dividing T cells have phenotypes similar to those activated by cognate antigens. The rapid homeostatic proliferation is caused by a lack of CD24 on dendritic cells (DCs). Interestingly, although CD24 expression in T cells is required for optimal homeostatic proliferation in the wild-type (WT) host, mice lacking CD24 on all cell types still mount higher homeostatic proliferation than the WT mice. Thus, a lack of CD24 in the non–T host cells bypassed the requirement for T cell expression of CD24 in homeostatic proliferation in the WT host. Our data demonstrate that CD24 expressed on the DCs limits T cell response to homeostatic cue and prevents fatal damage associated with uncontrolled homeostatic proliferation

The EGFR tyrosine kinase inhibitors as second-line therapy for EGFR wild-type non-small-cell lung cancer: a real-world study in People's Republic of China

INTRODUCTION: Clinical evidence comparing chemotherapy and tyrosine kinase inhibitors (TKIs) as second-line therapy for epidermal growth factor receptor (EGFR) wild-type non-small-cell lung cancer (NSCLC) are conflicting. METHODS: We retrospectively reviewed stage IV EGFR wild-type NSCLC patients who relapsed on first-line chemotherapy at the Shanghai Chest Hospital to compare the efficacy of TKIs and chemotherapy as second-line therapy among different clinical subgroups. RESULTS: The progression-free survival (PFS) and overall survival for patients receiving chemotherapy as second-line therapy for NSCLC were longer than patients who received TKIs. The hazard ratios (HRs) were 0.40 (P<0.001) and 0.50 (P<0.001), respectively. Subgroup analyses showed that second-line TKI therapy resulted in inferior PFS among smokers (HR =0.24, P<0.001), males (HR =0.33, P<0.001), females (HR =0.54, P=0.004), and patients with adenocarcinoma (HR =0.48, P<0.001) and nonadenocarcinoma histology (HR =0.20, P<0.001). Among never-smokers, the PFS in cohorts receiving second-line chemotherapy or TKIs was not significantly different (HR =0.70, P=0.08). CONCLUSION: These results suggest that EGFR TKI therapy was inferior compared to chemotherapy in EGFR wild-type NSCLC patients who relapsed from first-line chemotherapy; however, among never-smokers, these two treatment strategies were comparable