Systematic mapping of TF-mediated cell fate changes by a pooled induction coupled with scRNA-seq and multi-omics approaches.

Transcriptional regulation controls cellular functions through interactions between transcription factors (TFs) and their chromosomal targets. However, understanding the fate conversion potential of multiple TFs in an inducible manner remains limited. Here, we introduce iTF-seq as a method for identifying individual TFs that can alter cell fate toward specific lineages at a single-cell level. iTF-seq enables time course monitoring of transcriptome changes, and with biotinylated individual TFs, it provides a multi-omics approach to understanding the mechanisms behind TF-mediated cell fate changes. Our iTF-seq study in mouse embryonic stem cells identified multiple TFs that trigger rapid transcriptome changes indicative of differentiation within a day of induction. Moreover, cells expressing these potent TFs often show a slower cell cycle and increased cell death. Further analysis using bioChIP-seq revealed that GCM1 and OTX2 act as pioneer factors and activators by increasing gene accessibility and activating the expression of lineage specification genes during cell fate conversion. iTF-seq has utility in both mapping cell fate conversion and understanding cell fate conversion mechanisms.

Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.

Dynamics of optical injection in an external cavity based FP-LD for wide tunable microwave signal generation.

In this paper, we analyze the dynamics of optical injection in an external cavity based Fabry-Pérot laser diode (ECFP-LD) for wide tunable microwave signal generation. The ECFP-LD is a specially designed FP-LD that has a self-locked single dominant mode. The injected beam power is varied to analyze the dynamics of optical beam injection on the ECFP-LD. The ECFP-LD shows the interesting behavior of red-shift followed by hopping to another self-injected mode equivalent to FP-LD external and internal cavity modes separation, which provides the fine and coarse tuning of the self-injected mode. The optical beating of the injected beam and the self-injected mode, whether it is the fine red-shifted self-injected mode or the hopped self-injected mode equivalent to the external or internal cavity mode separation, provides a wide tunable range of microwave generation. We obtained fine tuning of 3 GHz for every self-injected mode, and coarse tuning of 15 GHz and 150 GHz, which is equivalent to the free spacing tuning of the external cavity (0.12 nm) and internal FP-LD cavity (1.17 nm), with change in the power of the injected beam. The maximum coarse tuning range is about 3.72 nm, and the corresponding beating frequency tuning range is 330 GHz. Hence, a wide tunable microwave frequency can be obtained by optical beating of the shifted/hopped self-injected mode and the injected beam.

TRRUST v2: an expanded reference database of human and mouse transcriptional regulatory interactions.

Transcription factors (TFs) are major trans-acting factors in transcriptional regulation. Therefore, elucidating TF-target interactions is a key step toward understanding the regulatory circuitry underlying complex traits such as human diseases. We previously published a reference TF-target interaction database for humans-TRRUST (Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining)-which was constructed using sentence-based text mining, followed by manual curation. Here, we present TRRUST v2 (www.grnpedia.org/trrust) with a significant improvement from the previous version, including a significantly increased size of the database consisting of 8444 regulatory interactions for 800 TFs in humans. More importantly, TRRUST v2 also contains a database for TF-target interactions in mice, including 6552 TF-target interactions for 828 mouse TFs. TRRUST v2 is also substantially more comprehensive and less biased than other TF-target interaction databases. We also improved the web interface, which now enables prioritization of key TFs for a physiological condition depicted by a set of user-input transcriptional responsive genes. With the significant expansion in the database size and inclusion of the new web tool for TF prioritization, we believe that TRRUST v2 will be a versatile database for the study of the transcriptional regulation involved in human diseases.

Depth plane adaptive integral imaging using a varifocal liquid lens array.

This paper proposes an enhanced integral imaging system with an electrically controllable image plane to address the issue of the limited depth problem in integral imaging. For implementation of the variable image plane, a varifocal liquid lens array and driving device are adopted instead of an ordinary solid lens array. The position of the central depth plane is varied by adjusting the focal length of the lens array. The proposed system enables matching between the object position and depth plane electrically, and thus an object moving from 5.15 to 11.72 cm is clearly displayed with this method.

Mechanical Rejuvenation of Mesenchymal Stem Cells from Aged Patients.

Mesenchymal stem cells (MSC) are an appealing therapeutic cell type for many diseases. However, patients with poor health or advanced age often have MSCs with poor regenerative properties. A major limiter of MSC therapies is cellular senescence, which is marked by limited proliferation capability, diminished multipotency, and reduced regenerative properties. In this work, we explored the ability of applied mechanical forces to reduce cellular senescence in MSCs. Our studies revealed that mechanical conditioning caused a lasting enhancement in proliferation, overall cell culture expansion potential, multipotency, and a reduction of senescence in MSCs from aged donors. Mechanistic studies suggested that these functional enhancements were mediated by oxidative stress and DNA damage repair signaling with mechanical load altering the expression of proteins of the sirtuin pathway, the DNA damage repair protein ATM, and antioxidant proteins. In addition, our results suggest a biophysical mechanism in which mechanical stretch leads to improved recognition of damaged DNA in the nucleus. Analysis of the cells through RNA-seq and ATAC-seq, demonstrated that mechanical loading alters the cell's genetic landscape to cause broad shifts in transcriptomic patterns that related to senescence. Overall, our results demonstrate that mechanical conditioning can rejuvenate mesenchymal stem cells derived from aged patients and improve their potential as a therapeutic cell type.

The transcriptional regulatory network modulating human trophoblast stem cells to extravillous trophoblast differentiation.

During human pregnancy, extravillous trophoblasts play crucial roles in placental invasion into the maternal decidua and spiral artery remodeling. However, regulatory factors and their action mechanisms modulating human extravillous trophoblast specification have been unknown. By analyzing dynamic changes in transcriptome and enhancer profile during human trophoblast stem cell to extravillous trophoblast differentiation, we define stage-specific regulators, including an early-stage transcription factor, TFAP2C, and multiple late-stage transcription factors. Loss-of-function studies confirm the requirement of all transcription factors identified for adequate differentiation, and we reveal that the dynamic changes in the levels of TFAP2C are essential. Notably, TFAP2C pre-occupies the regulatory elements of the inactive extravillous trophoblast-active genes during the early stage of differentiation, and the late-stage transcription factors directly activate extravillous trophoblast-active genes, including themselves as differentiation further progresses, suggesting sequential actions of transcription factors assuring differentiation. Our results reveal stage-specific transcription factors and their inter-connected regulatory mechanisms modulating extravillous trophoblast differentiation, providing a framework for understanding early human placentation and placenta-related complications.

Ancient eukaryotic protein interactions illuminate modern genetic traits and disorders.

All eukaryotes share a common ancestor from roughly 1.5 - 1.8 billion years ago, a single-celled, swimming microbe known as LECA, the Last Eukaryotic Common Ancestor. Nearly half of the genes in modern eukaryotes were present in LECA, and many current genetic diseases and traits stem from these ancient molecular systems. To better understand these systems, we compared genes across modern organisms and identified a core set of 10,092 shared protein-coding gene families likely present in LECA, a quarter of which are uncharacterized. We then integrated >26,000 mass spectrometry proteomics analyses from 31 species to infer how these proteins interact in higher-order complexes. The resulting interactome describes the biochemical organization of LECA, revealing both known and new assemblies. We analyzed these ancient protein interactions to find new human gene-disease relationships for bone density and congenital birth defects, demonstrating the value of ancestral protein interactions for guiding functional genetics today.

Human reference gut microbiome catalog including newly assembled genomes from under-represented Asian metagenomes.

BACKGROUND: Metagenome sampling bias for geographical location and lifestyle is partially responsible for the incomplete catalog of reference genomes of gut microbial species. Thus, genome assembly from currently under-represented populations may effectively expand the reference gut microbiome and improve taxonomic and functional profiling. METHODS: We assembled genomes using public whole-metagenomic shotgun sequencing (WMS) data for 110 and 645 fecal samples from India and Japan, respectively. In addition, we assembled genomes from newly generated WMS data for 90 fecal samples collected from Korea. Expecting genome assembly for low-abundance species may require a much deeper sequencing than that usually employed, so we performed ultra-deep WMS (> 30 Gbp or > 100 million read pairs) for the fecal samples from Korea. We consequently assembled 29,082 prokaryotic genomes from 845 fecal metagenomes for the three under-represented Asian countries and combined them with the Unified Human Gastrointestinal Genome (UHGG) to generate an expanded catalog, the Human Reference Gut Microbiome (HRGM). RESULTS: HRGM contains 232,098 non-redundant genomes for 5414 representative prokaryotic species including 780 that are novel, > 103 million unique proteins, and > 274 million single-nucleotide variants. This is an over 10% increase from the UHGG. The new 780 species were enriched for the Bacteroidaceae family, including species associated with high-fiber and seaweed-rich diets. Single-nucleotide variant density was positively associated with the speciation rate of gut commensals. We found that ultra-deep sequencing facilitated the assembly of genomes for low-abundance taxa, and deep sequencing (e.g., > 20 million read pairs) may be needed for the profiling of low-abundance taxa. Importantly, the HRGM significantly improved the taxonomic and functional classification of sequencing reads from fecal samples. Finally, analysis of human self-antigen homologs on the HRGM species genomes suggested that bacterial taxa with high cross-reactivity potential may contribute more to the pathogenesis of gut microbiome-associated diseases than those with low cross-reactivity potential by promoting inflammatory condition. CONCLUSIONS: By including gut metagenomes from previously under-represented Asian countries, Korea, India, and Japan, we developed a substantially expanded microbiome catalog, HRGM. Information of the microbial genomes and coding genes is publicly available ( www.mbiomenet.org/HRGM/ ). HRGM will facilitate the identification and functional analysis of disease-associated gut microbiota.

A Genome-Scale Co-Functional Network of Xanthomonas Genes Can Accurately Reconstruct Regulatory Circuits Controlled by Two-Component Signaling Systems.

Bacterial species in the genus Xanthomonas infect virtually all crop plants. Although many genes involved in Xanthomonas virulence have been identified through molecular and cellular studies, the elucidation of virulence-associated regulatory circuits is still far from complete. Functional gene networks have proven useful in generating hypotheses for genetic factors of biological processes in various species. Here, we present a genome-scale co-functional network of Xanthomonas oryze pv. oryzae (Xoo) genes, XooNet (www.inetbio.org/xoonet/), constructed by integrating heterogeneous types of genomics data derived from Xoo and other bacterial species. XooNet contains 106,000 functional links, which cover approximately 83% of the coding genome. XooNet is highly predictive for diverse biological processes in Xoo and can accurately reconstruct cellular pathways regulated by two-component signaling transduction systems (TCS). XooNet will be a useful in silico research platform for genetic dissection of virulence pathways in Xoo.

Network Integrative Genomic and Transcriptomic Analysis of Carbapenem-Resistant Klebsiella pneumoniae Strains Identifies Genes for Antibiotic Resistance and Virulence.

Global increases in the use of carbapenems have resulted in several strains of Gram-negative bacteria acquiring carbapenem resistance, thereby limiting treatment options. Klebsiella pneumoniae is a common carbapenem-resistant pathogenic bacterium that is widely studied to identify novel antibiotic resistance mechanisms and drug targets. Antibiotic-resistant clinical isolates generally harbor many genetic alterations, and the identification of responsible mutations would provide insights into the molecular mechanisms of antibiotic resistance. We propose a method to prioritize mutated genes responsible for antibiotic resistance on the basis of expression changes in their local subnetworks, hypothesizing that mutated genes that show significant expression changes among the corresponding functionally associated genes are more likely to be involved in the carbapenem resistance. For network-based gene prioritization, we developed KlebNet (www.inetbio.org/klebnet), a genome-scale cofunctional network of K. pneumoniae genes. Using KlebNet, we reconstructed the functional modules for carbapenem resistance and virulence and identified the functional association between antibiotic resistance and virulence. Using complementation assays with the top candidate genes, we were able to validate a novel gene that negatively regulated carbapenem resistance and four novel genes that positively regulated virulence in Galleria mellonella larvae. Therefore, our study demonstrated the feasibility of network-based identification of genes required for antibiotic resistance and virulence of human-pathogenic bacteria.IMPORTANCE Klebsiella pneumoniae is a major bacterial pathogen that causes pneumonia and urinary tract infections in human. K. pneumoniae infections are treated with carbapenem, but carbapenem-resistant K. pneumoniae has been spreading worldwide. We are able to identify antimicrobial-resistant genes among mutated genes of the antibiotic-resistant clinical isolates. However, they usually harbor many mutated genes, including those that cause weak or neutral functional effects. Therefore, we need to prioritize the mutated genes to identify the more likely candidates for the follow-up functional analysis. For this study, we present a functional network of K. pneumoniae genes and propose a network-based method of prioritizing the mutated genes of the resistant clinical isolates. We also reconstructed the network-based functional modules for carbapenem resistance and virulence and retrieved the functional association between antibiotic resistance and virulence. This study demonstrated the feasibility of network-based analysis of clinical genomics data for the study of K. pneumoniae infection.

Intraoperative burn from a grounding pad of electrosurgical device during breast surgery: A CARE-compliant case report.

RATIONAL: Burns at the site of the return electrode (i.e., grounding pad) are possible effects of electrosurgery. Despite this knowledge, however, ignorance or negligence with regards to proper handling of the grounding pads still often occurs. Burn injuries can be easily prevented by taking the necessary precautions; thus, during plastic surgery, careful attention should to be paid. PATIENT CONCERNS: A 38-year-old female patient was admitted to our ward to be performed augmentation mammoplasty. Before the start of the procedure, the grounding pad was placed on the surgical table and the left calf of the patient was placed on the grounding pad. Before using the endoscope, we found a burn on patient's left calf, where the grounding pad had been placed. DIAGNOSIS: It was a 3-cm-by-3-cm-sized full thickness burn. The surrounding areas had no painful sensation with noninfectious sign. INTERVENTION: Debridement and direct closure was performed with elliptical incision of eschar. OUTCOMES: The patient did not require additional surgical procedure anymore and satisfied with the scar. LESSONS: Through this case, we present the appropriate management of electrical burns from a grounding pad, and emphasize the understanding of the mechanism of burn because of electrosurgery, and how to use the grounding pad optimally to minimize the patient's risk.

Network-based genetic investigation of virulence-associated phenotypes in methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a gram-positive bacterium that causes a wide range of infections. Recently, the spread of methicillin-resistant S. aureus (MRSA) strains has seriously reduced antibiotic treatment options. Anti-virulence strategies, the objective of which is to target the virulence instead of the viability of the pathogen, have become widely accepted as a means of avoiding the emergence of new antibiotic-resistant strains. To increase the number of anti-virulence therapeutic options, it is necessary to identify as many novel virulence-associated genes as possible in MRSA. Co-functional networks have proved useful for mapping gene-to-phenotype associations in various organisms. Herein, we present StaphNet (www.inetbio.org/staphnet), a genome-scale co-functional network for an MRSA strain, S. aureus subsp. USA300_FPR3757. StaphNet, which was constructed by the integration of seven distinct types of genomics data within a Bayesian statistics framework, covers approximately 94% of the coding genome with a high degree of accuracy. We implemented a companion web server for network-based gene prioritization of the phenotypes of 31 different S. aureus strains. We demonstrated that StaphNet can effectively identify genes for virulence-associated phenotypes in MRSA. These results suggest that StaphNet can facilitate target discovery for the development of anti-virulence drugs to treat MRSA infection.

N-type graphene induced by dissociative H2 adsorption at room temperature.

Studies of the interaction between hydrogen and graphene have been increasingly required due to the indispensable modulation of the electronic structure of graphene for device applications and the possibility of using graphene as a hydrogen storage material. Here, we report on the behaviour of molecular hydrogen on graphene using the gate voltage-dependent resistance of single-, bi-, and multi-layer graphene sheets as a function of H2 gas pressure up to 24 bar from 300 K to 345 K. Upon H2 exposure, the charge neutrality point shifts toward the negative gate voltage region, indicating n-type doping, and distinct Raman signature changes, increases in the interlayer distance of multi-layer graphene, and a decrease in the d-spacing occur, as determined by TEM. These results demonstrate the occurrence of dissociative H2 adsorption due to the existence of vacancy defects on graphene.