bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential
Authors: Martinez David R. , Moreira Fernando R. , Zweigart Mark R. , Gully Kendra L. , Cruz Gabriela De la , Brown Ariane J. , Adams Lily E. , Catanzaro Nicholas , Yount Boyd , Baric Thomas J. , Mallory Michael L. , Conrad Helen , May Samantha R. , Dong Stephanie , Scobey D. Trevor , Montgomery Stephanie A. , Perry Jason , Babusis Darius , Barrett Kimberly T. , Nguyen Anh-Hoa , Nguyen Anh-Quan , Kalla Rao , Bannister Roy , Bilello John P. , Feng Joy Y. , Cihlar Tomas , Baric Ralph S. , Mackman Richard L. , Schäfer Alexandra , Sheahan Timothy P.
Published: June 2023

Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.
Identifying molecular targets of a colloidal nanosilver formulation (Silversol®) in multidrug resistantPseudomonas aeruginosa
Authors: Gajera Gemini , Thakkar Nidhi , Godse Chhaya , DeSouza Anselm , Mehta Dilip , Kothari Vijay
Published: June 2023

P. aeruginosais a notorious pathogen. A multi-drug resistant strain of this bacterium was challenged with a colloidal nano-silver formulation- Silversol®. Its minimum inhibitory concentration againstP. aeruginosawas found to be 1.5 ppm, and at sub-MIC of 1 ppm, it was able to alter quorum-sensing regulated pigmentation, exopolysaccharide synthesis and biofilm formation, antibiotic susceptibility, protein synthesis and export, nitrogen metabolism, and siderophore production in this pathogen. Transcriptome analysis of the silver-exposedP. aeruginosaindicated generation of nitrosative stress and disturbance of iron homeostasis to be the major mechanisms associated with anti-Pseudomonas activity of Silversol®. Network analysis of the differentially expressed genes in silver-treated bacterium identified ten genes as the potential molecular targets: norB, norD, nirS, nirF, nirM, nirQ, nosZ, nosY, narK1, and norE (all associated with nitrogen metabolism or denitrification). Three of them (norB, narK1, and norE) were also validated through RT-PCR.
Differences in Relevant Physicochemical Properties Correlate with Synergistic Activity of Antimicrobial Peptides
Authors: Medvedeva Angela , Teimouri Hamid , Kolomeisky Anatoly B.
Published: June 2023

With the urgent need for new medical approaches due to increased bacterial resistance to antibiotics, antimicrobial peptides (AMPs) have been considered as potential treatments for infections. Experiments indicate that combinations of several types of AMPs might be more effective at inhibiting bacterial growth with reduced toxicity and a lower likelihood of inducing bacteria resistance. The molecular mechanisms of AMP-AMP synergistic antimicrobial activity, however, remain not well understood. Here, we present a theoretical approach that allows us to relate the physicochemical properties of AMPs and their antimicrobial cooperativity. A concept of physicochemical similarity is introduced, and it is found that less similar AMPs with respect to certain physicochemical properties lead to greater synergy because of their complementary antibacterial actions. The analysis of correlations between the similarity and the antimicrobial properties allows us to effectively separate synergistic from non-synergistic AMPs pairs. Our theoretical approach can be used for the rational design of more effective AMPs combinations for specific bacterial targets, for clarifying the mechanisms of bacterial elimination, and for a better understanding of cooperativity phenomena in biological systems.

Author summary

It is impossible to imagine modern medicine without antibiotics. But there is a growing problem of increased bacterial resistance to them. These considerations stimulated a search for novel methods to defend against infections. Antimicrobial peptides (AMPs) came out as powerful antibacterial agents. It was also found that combinations of AMPs are even more efficient than individual peptides. The mechanisms of such synergistic activities, however, are not understood. We developed a computational framework that allows us to connect the physicochemical properties of AMPs and their abilities to cooperatively eliminate infections. It is found that less similar peptides might exhibit synergy because of their complementary antibacterial properties. Our theoretical approach might lead to a better rational design of new antimicrobial drugs.
Appropriate sampling and long follow-up are required to rigorously evaluate longevity of humoral memory after vaccination
Authors: Ganusov Vitaly V.
Published: June 2023

One of the goals of vaccination is to induce long-term immunity against the infection and/or disease. However, evaluating the duration of protection following vaccination often requires long-term follow-ups that can conflict with the desire to rapidly publish results. Arunachalam et al. JCI 2023 followed individuals receiving third or fourth dose of mRNA COVID19 vaccines for up to 6 months and in finding that the levels of SARS-CoV2-specific antibodies (Abs) declined with similar rates for the two groups came to the conclusion that additional boosting is unnecessary to prolong immunity to SARS-CoV-2. However, this may be premature conclusion to make. Accordingly, we demonstrate that measuring Ab levels at 3 time points and only for a short (up to 6 month) duration does not allow to accurately and rigorously evaluate the long-term half-life of vaccine-induced Abs. By using the data from a cohort of blood donors followed for several years, we show that after re-vaccination with vaccinia virus (VV), VV-specific Abs decay bi-phasically and even the late decay rate exceeds the true slow loss rate of humoral memory observed years prior to the boosting. We argue that mathematical modeling should be used to better optimize sampling schedules to provide more reliable advice about the duration of humoral immunity after repeated vaccinations.
Rare variants in long non-coding RNAs are associated with blood lipid levels in the TOPMed Whole Genome Sequencing Study
Authors: Wang Yuxuan , Selvaraj Margaret Sunitha , Li Xihao , Li Zilin , Holdcraft Jacob A. , Arnett Donna K. , Bis Joshua C. , Blangero John , Boerwinkle Eric , Bowden Donald W. , Cade Brian E. , Carlson Jenna C. , Carson April P. , Chen Yii-Der Ida , Curran Joanne E. , de Vries Paul S. , Dutcher Susan K. , Ellinor Patrick T. , Floyd James S. , Fornage Myriam , Freedman Barry I. , Gabriel Stacey , Germer Soren , Gibbs Richard A. , Guo Xiuqing , He Jiang , Heard-Costa Nancy , Hildalgo Bertha , Hou Lifang , Irvin Marguerite R. , Joehanes Roby , Kaplan Robert C. , Kardia Sharon LR. , Kelly Tanika N. , Kim Ryan , Kooperberg Charles , Kral Brian G. , Levy Daniel , Li Changwei , Liu Chunyu , Lloyd-Jone Don , Loos Ruth JF. , Mahaney Michael C. , Martin Lisa W. , Mathias Rasika A. , Minster Ryan L. , Mitchell Braxton D. , Montasser May E. , Morrison Alanna C. , Murabito Joanne M. , Naseri Take , O’Connell Jeffrey R. , Palmer Nicholette D. , Preuss Michael H. , Psaty Bruce M. , Raffield Laura M. , Rao Dabeeru C. , Redline Susan , Reiner Alexander P. , Rich Stephen S. , Ruepena Muagututi’a Sefuiva , Sheu Wayne H-H. , Smith Jennifer A. , Smith Albert , Tiwari Hemant K. , Tsai Michael Y. , Viaud-Martinez Karine A. , Wang Zhe , Yanek Lisa R. , Zhao Wei ,   NHLBI Trans-Omics for Precision Medicine (TOPMed) Consortium, Rotter Jerome I. , Lin Xihong , Natarajan Pradeep , Peloso Gina M.
Published: June 2023

Long non-coding RNAs (lncRNAs) are known to perform important regulatory functions. Large-scale whole genome sequencing (WGS) studies and new statistical methods for variant set tests now provide an opportunity to assess the associations between rare variants in lncRNA genes and complex traits across the genome. In this study, we used high-coverage WGS from 66,329 participants of diverse ancestries with blood lipid levels (LDL-C, HDL-C, TC, and TG) in the National Heart, Lung, and Blood Institute (NHLBI) Trans-Omics for Precision Medicine (TOPMed) program to investigate the role of lncRNAs in lipid variability. We aggregated rare variants for 165,375 lncRNA genes based on their genomic locations and conducted rare variant aggregate association tests using the STAAR (variant-Set Test for Association using Annotation infoRmation) framework. We performed STAAR conditional analysis adjusting for common variants in known lipid GWAS loci and rare coding variants in nearby protein coding genes. Our analyses revealed 83 rare lncRNA variant sets significantly associated with blood lipid levels, all of which were located in known lipid GWAS loci (in a ±500 kb window of a Global Lipids Genetics Consortium index variant). Notably, 61 out of 83 signals (73%) were conditionally independent of common regulatory variations and rare protein coding variations at the same loci. We replicated 34 out of 61 (56%) conditionally independent associations using the independent UK Biobank WGS data. Our results expand the genetic architecture of blood lipids to rare variants in lncRNA, implicating new therapeutic opportunities.
ORBIT forE. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency
Authors: Saunders Scott H. , Ahmed Ayesha M.
Published: June 2023

Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput applications. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered inMycobacteria, and here we adapt and expand it forE. coli. Our redesigned plasmid toolkit achieved nearly 1000x higher efficiency than λ Red recombination and enabled precise, stable knockouts (<134 kb) and integrations (<11 kb) of various sizes. Additionally, we constructed multi-mutants (double and triple) in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT forE. coliis a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.
Enabling AI in Synthetic Biology through Construction File Specification
Authors: Ataii Nassim , Bakshi Sanjyot , Chen Yisheng , Fernandez Michael , Shao Zihang , Scheftel Zachary , Tou Connor , Vega Mia , Wang Yuting , Zhang Hanxiao , Zhao Zexuan , Anderson J. Christopher
Published: June 2023

The Construction File (CF) specification establishes a standardized interface for molecular biology operations, laying a foundation for automation and enhanced efficiency in experiment design. It is implemented across three distinct software projects: PyDNA_CF_Simulator, a Python project featuring a ChatGPT plugin for interactive parsing and simulating experiments; ConstructionFileSimulator, a field-tested Java project that showcases ‘Experiment’ objects expressed as flat files; and C6-Tools, a JavaScript project integrated with Google Sheets via Apps Script, providing a user-friendly interface for authoring and simulation of CF. The CF specification not only standardizes and modularizes molecular biology operations but also promotes collaboration, automation, and reuse, significantly reducing potential errors. The potential integration of CF with artificial intelligence, particularly GPT-4, suggests innovative automation strategies for synthetic biology. While challenges such as token limits, data storage, and biosecurity remain, proposed solutions promise a way forward in harnessing AI for experiment design. This shift from human-driven design to AI-assisted workflows, steered by high-level objectives, charts a potential future path in synthetic biology, envisioning an environment where complexities are managed more effectively.
Enteropathway: the metabolic pathway database for the human gut microbiota
Authors: Shiroma Hirotsugu , Darzi Youssef , Terajima Etsuko , Nakagawa Zenichi , Tsuchikura Hirotaka , Tsukuda Naoki , Moriya Yuki , Okuda Shujiro , Goto Susumu , Yamada Takuji
Published: June 2023

The human gut microbiota produces diverse, extensive metabolites which have the potential to affect host physiology. Despite significant efforts to identify metabolic pathways for producing these microbial metabolites, a comprehensive metabolic pathway database for the human gut microbiota is still lacking. Here, we present Enteropathway, a metabolic pathway database that integrates 3,121 compounds, 3,460 reactions, and 837 modules that were obtained from 835 manually curated scientific literature. Notably, 757 modules of these modules are new entries and cannot be found in any other databases. The database is accessible from a web application (https://enteropathway.org) that offers a metabolic diagram for graphical visualization of metabolic pathways, a customization interface, and an enrichment analysis feature for highlighting enriched modules on the metabolic diagram. Overall, Enteropathway is a comprehensive reference database and a tool for visual and statistical analysis in human gut microbiota studies and was designed to help researchers pinpoint new insights into the complex interplay between microbiota and host metabolism.
The alternative sigma factor RpoD4 pulses at division and links the clock and the cell cycle inSynechococcus elongatus
Authors: Ye Chao , Saez Teresa , Das Arijit K. , Martins Bruno M.C. , Locke James C.W.
Published: June 2023

The cyanobacterial circadian clock is an extensive regulator of gene expression, generating a 24-hour rhythm in the majority of genes inSynechococcus elongatusPCC7942. This raises the question of how the cyanobacterial clock regulates, and receives input from, the diverse cellular processes it controls. Alternative sigma factors, which bind to the core RNA polymerase enzyme and direct the synthesis of specific sets of transcripts, are one mechanism for the clock to set the time of its outputs. In this work, we use single-cell time-lapse microscopy to reveal the transcriptional dynamics of RpoD4, an alternative sigma factor that was previously reported as either arrhythmic or circadian. We find instead that RpoD4 pulses at cell division, dynamics missed by previous bulk averaging. The circadian clock modulates the amplitude of RpoD4 expression pulses, as well as the timing of pulses through its control of division timing. In turn, arpoD4mutation causes a reduction in clock period. Further, arpoD4mutant results in smaller cell size, and increasing expression of RpoD4 results in larger cell sizes in a dose-response manner, allowing tunable control of cell size. Thus, our single-cell analysis has revealed pulsing gene expression dynamics in cyanobacteria, linking the clock, RpoD4 and the cell cycle.
Electron microscopic and crystallographic studies of bacteriophage Sf6 procapsid-like particles assembled from heterologously expressed capsid protein gp5
Authors: Zhao Haiyan , Tang Liang
Published: June 2023

Many double-stranded DNA (dsDNA) viruses undergo a capsid maturation process during assembly of infectious virus particles, which involves transformation of a metastable capsid precursor called procapsid into a stable, DNA-filled capsid usually with a larger size and a more angular shape. Sf6 is a tailed dsDNA bacteriophage that infectsShigella flexneri. The phage Sf6 capsid protein gp5 was heterologously expressed and purified. Electron microscopy showed that the gp5 spontaneously assembled into spherical, procapsid-like particles. We also observed tube-like and cone-shaped particles reminiscent of human immunodeficiency virus. The gp5 procapsid-like particles were crystallized and crystals diffracted beyond 4.3 Å resolution. X-ray data at 5.9 Å resolution were collected with a completeness of 31.1% and an overallRmerge of 15.0%. The crystals belong to the space groupC2 with unit cell dimensions of a=973.326 Å, b=568.234 Å, c=565.567 Å, and β=120.540°. Self-rotation function showed the 532 symmetry, confirming formation of icosahedral particles. The particle was situated at the origin of the crystal unit cell with the icosahedral 2-fold axis coinciding with the crystallographicbaxis, and there is a half of the icosahedral particle in the crystallographic asymmetric unit.
Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility
Authors: Böhringer David , Bauer Andreas , Moravec Ivana , Bischof Lars , Kah Delf , Mark Christoph , Grundy Thomas J. , Görlach Ekkehard , O’Neill Geraldine M , Budday Silvia , Strissel Pamela , Strick Reiner , Malandrino Andrea , Gerum Richard , Mak Michael , Rausch Martin , Fabry Ben
Published: June 2023

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements severely limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) Inhibitor-induced changes of traction forces and fiber alignment and density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.
Liver-specification of human iPSC-derived endothelial cells transplanted into mouse liver
Authors: Yap Kiryu K. , Schröder Jan , Gerrand Yi-Wen , Kong Anne M. , Fox Adrian M. , Knowles Brett , Banting Simon W. , Elefanty Andrew G. , Stanley Eduoard G. , Yeoh George C. , Lockwood Glen P. , Cogger Victoria C. , Morrison Wayne A. , Polo Jose M. , Mitchell Geraldine M.
Published: June 2023

Liver sinusoidal endothelial cells (LSECs) play an important role in liver development, regeneration and pathophysiology, but the differentiation process that generates their unique tissue-specific phenotype is poorly understood and difficult to study as primary cells are only available in limited quantities. To address this, we hypothesised that human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) can produce hiPSC-derived LSECs upon transplantation into the livers ofFah−/−/Rag2−/−/Il2rg−/−mice, and serve as a model to study LSEC specification. Progressive and long-term repopulation of the liver vasculature was observed, as iECs expanded along the sinusoids that run between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptome of transplanted cells at time-points between 1 and 12 weeks were compared against primary human adult LSECs, which demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factorsNOTCH1,GATA4, andFOSplay a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process includeEMCNandCLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contain zonal subpopulations with a region-specific phenotype. Collectively, this study confirms that hiPSC can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs.
Serovar-level Identification of Bacterial Foodborne Pathogens From Full-length 16S rRNA Gene Sequencing
Authors: Grinevich Dmitry , Harden Lyndy , Thakur Siddhartha , Callahan Benjamin J
Published: June 2023

The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogensEscherichia coliandSalmonella, the primary sub-species classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are therefore of interest for pathogen surveillance. Here we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotypingS. entericaandE. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predictingSalmonellaserotypes onin silicotest data, and identified key pathogenic serovars ofSalmonellaandE. coliin isolate and environmental test samples. Although serotype prediction from 16S sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intra-species variation and direct sequencing from environmental samples could be valuable.
Targeting Ribosome Biogenesis as a Novel Therapeutic Approach to Overcome EMT-related Chemoresistance in Breast Cancer
Authors: Ban Yi , Zou Yue , Liu Yingzhuo , Lee Sharrell B. , Bednarczyk Robert B. , Sheng Jianting , Cao Yuliang , Wong Stephen T. C. , Gao Dingcheng
Published: June 2023

Epithelial-to-mesenchymal transition (EMT) contributes significantly to chemotherapy resistance and remains a critical challenge in treating advanced breast cancer. The complexity of EMT, involving redundant pro-EMT signaling pathways and its paradox reversal process, mesenchymal-to-epithelial transition (MET), has hindered the development of effective treatments. In this study, we utilized a Tri-PyMT EMT lineage-tracing model and single-cell RNA sequencing (scRNA-seq) to comprehensively analyze the EMT status of tumor cells. Our findings revealed elevated ribosome biogenesis (RiBi) during the transitioning phases of both EMT and MET processes. RiBi and its subsequent nascent protein synthesis mediated by ERK and mTOR signalings are essential for EMT/MET completion. Importantly, inhibiting excessive RiBi genetically or pharmacologically impaired the EMT/MET capability of tumor cells. Combining RiBi inhibition with chemotherapy drugs synergistically reduced metastatic outgrowth of epithelial and mesenchymal tumor cells under chemotherapies. Our study suggests that targeting the RiBi pathway presents a promising strategy for treating patients with advanced breast cancer.

Significance

This study uncovers the crucial involvement of ribosome biogenesis (RiBi) in the regulation of epithelial and mesenchymal state oscillations in breast cancer cells, which plays a major role in the development of chemoresistant metastasis. By proposing a novel therapeutic strategy targeting the RiBi pathway, the study offers significant potential to enhance treatment efficacy and outcomes for patients with advanced breast cancer. This approach could help overcome the limitations of current chemotherapy options and address the complex challenges posed by EMT-mediated chemoresistance.
Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies
Authors: Husser Mathieu C. , Pham Nhat P. , Law Chris , Araujo Flavia R. B. , Martin Vincent J.J. , Piekny Alisa
Published: June 2023

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.
Lanthanide-dependent isolation of phyllosphere methylotrophs selects for a phylogenetically conserved but metabolically diverse community
Authors: Govindaraju Alekhya M. , Friel Colleen A. , Good Nathan M. , Banks Sidney L. , Wayne Kenan S. , Martinez-Gomez N. Cecilia
Published: June 2023

The influence of lanthanide biochemistry during methylotrophy demands a reassessment of how the composition and metabolic potential of methylotrophic phyllosphere communities are affected by the presence of these metals. To investigate this, methylotrophs were isolated from soybean leaves by selecting for bacteria capable of methanol oxidation with lanthanide cofactors. Of the 344 pink-pigmented facultative methylotroph isolates, none were obligately lanthanide-dependent. Phylogenetic analyses revealed that all strains were nearly identical to each other and to model strains from theextorquensclade ofMethylobacterium, withrpoBproviding higher resolution than 16srRNA for strain-specific identification. Despite the low species diversity, the metabolic capabilities of the community diverged greatly. Strains encoding identical PQQ-dependent alcohol dehydrogenases displayed significantly different growth from each other on alcohols in the presence and absence of lanthanides. Several strains also lacked well-characterized lanthanide-associated genes thought to be important for phyllosphere colonization. Additionally, 3% of our isolates were capable of growth on sugars and 23% were capable of growth on aromatic acids, substantially expanding the range of multicarbon substrates utilized by members of theextorquensclade in the phyllosphere. Whole genome sequences of eleven novel strains are reported. Our findings suggest that the expansion of metabolic capabilities, as well as differential usage of lanthanides and their influence on metabolism among closely related strains, point to evolution of niche partitioning strategies to promote colonization of the phyllosphere.

Importance

Lanthanides are essential metals for life. The identification of lanthanide-associated processes has been well-studied in methylotrophic bacteria, which are plant symbionts that utilize reduced one-carbon compounds for growth. Yet, the importance of lanthanides in plant-microbe interactions and the effects of lanthanides on microbial physiology and colonization in plants remains poorly understood. Here, we characterize the first methylotrophic bacterial community isolated from the phyllosphere in a lanthanide-dependent manner. We have identified strains encoding identical lanthanide-dependent enzymes yet exhibiting differences in lanthanide-associated growth, and have identified strains lacking lanthanide-associated genes thought to be important for phyllosphere colonization. In addition, we have identified many strains capable of metabolisms that were thought to be rare within this clade. Overall, our isolates serve as a microcosm by which to interrogate how lanthanides influence methylotrophic physiology in plant environments and highlights how phylogenetically similar strains can diverge greatly in metabolic potential.
Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM
Authors: Wankowicz Stephanie A. , Ravikumar Ashraya , Sharma Shivani , Riley Blake T. , Raju Akshay , Flowers Jessica , Hogan Daniel , van den Bedem Henry , Keedy Daniel A. , Fraser James S.
Published: June 2023

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift towards modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfreeand geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g. Coot) and fit can be further improved by refinement using standard pipelines (e.g. Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.
A cellular and spatial atlas ofTP53-associated tissue remodeling in lung adenocarcinoma
Authors: Zhao William , Kepecs Benjamin , Mahadevan Navin R. , Segerstolpe Asa , Weirather Jason L. , Besson Naomi R. , Giotti Bruno , Soong Brian Y. , Li Chendi , Vigneau Sebastien , Slyper Michal , Wakiro Isaac , Jane-Valbuena Judit , Ashenberg Orr , Rotem Asaf , Bueno Raphael , Rozenblatt-Rosen Orit , Pfaff Kathleen , Rodig Scott , Hata Aaron N. , Regev Aviv , Johnson Bruce E. , Tsankov Alexander M.
Published: June 2023

TP53is the most frequently mutated gene across many cancers and is associated with shorter survival in lung adenocarcinoma (LUAD). To define howTP53mutations affect the LUAD tumor microenvironment (TME), we constructed a multi-omic cellular and spatial tumor atlas of 23 treatment-naïve human lung tumors. We found thatTP53-mutant (TP53mut) malignant cells lose alveolar identity and upregulate highly proliferative and entropic gene expression programs consistently across resectable LUAD patient tumors, genetically engineered mouse models, and cell lines harboring a wide spectrum ofTP53mutations. We further identified a multicellular tumor niche composed ofSPP1+macrophages and collagen-expressing fibroblasts that coincides with hypoxic, pro-metastatic expression programs inTP53muttumors. Spatially correlated angiostatic and immune checkpoint interactions, includingCD274-PDCD1andPVR-TIGIT, are also enriched inTP53mutLUAD tumors, which may influence response to checkpoint blockade therapy. Our methodology can be further applied to investigate mutation-specific TME changes in other cancers.
CoPheScan: phenome-wide association studies accounting for linkage disequilibrium
Authors: Manipur Ichcha , Reales Guillermo , Sul Jae Hoon , Shin Myung Kyun , Longerich Simonne , Cortes Adrian , Wallace Chris
Published: June 2023

Phenome-wide association studies (PheWAS) facilitate the discovery of associations between a single genetic variant with multiple phenotypes. For variants which impact a specific protein, this can help identify additional therapeutic indications or on-target side effects of intervening on that protein. However, PheWAS is restricted by an inability to distinguish confounding due to linkage disequilibrium (LD) from true pleiotropy. Here we describe CoPheScan (Coloc adapted Phenome-wide Scan), a Bayesian approach that enables an intuitive and systematic exploration of causal associations while simultaneously addressing LD confounding. We demonstrate its performance through simulation, showing considerably better control of false positive rates than a conventional approach not accounting for LD. We used CoPheScan to perform PheWAS of protein-truncating variants and fine-mapped variants from disease and pQTL studies, in 2275 disease phenotypes from the UK Biobank. Our results identify the complexity of known pleiotropic genes such asAPOE, and suggest a new causal role forTGM3in skin cancer.
Sse1, Hsp110 chaperone of yeast, controls the cellular fate during Endoplasmic Reticulum-stress
Authors: Jha Mainak Pratim , Kumar Vignesh , Sharma Lakshita , Ghosh Asmita , Mapa Koyeli
Published: June 2023

Sse1 is a cytosolic Hsp110 molecular chaperone of yeast,Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as Nucleotide Exchange Factor (NEF), as protein-disaggregase and as a Chaperone linked to Protein Synthesis (CLIPS), are well documented. In the currently study, we show thatSSE1genetically interacts withIRE1andHAC1, the Endoplasmic Reticulum-Unfolded Protein Response (ER-UPR) sensors implicating its role in ER protein homeostasis. Interestingly, absence of this chaperone imparts unusual resistance to tunicamycin-induced ER stress which depends on the intact Ire1-Hac1 mediated ER-UPR signalling. Furthermore, cells lackingSSE1show ER-stress-responsive inefficient reorganization of translating ribosomes from polysomes to monosomes and increased monosome content that drive uninterrupted protein translation. In consequence, the kinetics of ER-UPR is starkly different insse1Δ strain where we show that stress response induction and restoration of homeostasis is prominently faster in contrast to the wildtype (WT) cells. Importantly, Sse1 plays a critical role in controlling the ER-stress mediated cell division arrest which is escaped insse1Δ strain during chronic tunicamycin stress. Consequently,sse1Δ strain shows significantly higher cell viability in comparison to WT yeast, following short-term as well as long-term tunicamycin stress. In summary, we demonstrate a new role of Sse1 in ER protein homeostasis where the chaperone genetically interacts with ER-UPR pathway, controls the protein translation during ER stress and the kinetics of ER-UPR. More importantly, we show the crtical role of Sse1 in regulating the ER-stress-induced cell division arrest and cell death during global ER stress by tunicamycin.

Author Summary

Sse1 is a cytosolic Hsp110 molecular chaperone of yeast,Saccharomyces cerevisiae. It performs various functions as Nucleotide Exchange Factor (NEF), as a protein-disaggregase and as a Chaperone linked to Protein Synthesis (CLIPS) to maintain a healthy proteome in the cytosol. In the present study, we show thatSSE1is a critical player to maintain Endoplasmic Reticulum (ER) protein homeostasis. We report thatSSE1genetically interacts with the ER-Unfolded Protein Response (ER-UPR) sensors,IRE1andHAC1. In absence ofSSE1,yeast cells gain unusual fitness to tunicamycin (Tm)-induced ER stress which depends on the functional Ire1-Hac1 mediated ER-UPR signalling. Furthermore, Sse1 controls the ribosomal organization during tunicamycin-induced ER-stress and in absence of the chaperone the amount of polysomes and monosmes are high leading to uninterrupted protein translation during ER stress. Consequently,sse1Δ strain exhibits starkly different ER-UPR kinetics compared to wildtype (WT) cells. Importantly, our data reveal that cell division arrest and cell death due to Tm-induced ER-stress is critically controlled by Sse1.
Repurposing the dark genome. IV – noncoding proteins
Authors: Nayak Sarangdhar , Dhar Pawan K.
Published: June 2023

The dark genome comprising of non-expressing, non-translating, and extinct DNA sequences has remained a largely unexplored genomic space. Using computational and experimental approaches, novel insights into the dark matter genome have recently been gained, revealing the presence of a vast and unexplored resource. Non-coding RNA (ncRNA) refers to a class of RNA molecules that do not encode proteins but play important regulatory roles in the cell. We asked if it was possible to make functional peptides and proteins from ncRNA leading to a new biological insight and applications? Here we present initial computational data in support of making functional noncoding proteins (NCP) from ncRNA sequences. Different types of non-coding genomic sequences originating fromCaenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, andHomo sapienswere studied to understand sequence composition, secondary structure, and physiochemical properties of NCPs. This work builds the foundation for experimentally characterizing the first-in-the-class non-coding proteins leading to a novel insights and applications.
A SEVA-based, CRISPR-Cas3-assisted genome engineering approach forPseudomonaswith efficient vector curing
Authors: Lammens Eveline-Marie , Volke Daniel Christophe , Schroven Kaat , Voet Marleen , Kerremans Alison , Lavigne Rob , Hendrix Hanne
Published: June 2023

The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration inPseudomonas putida, an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system fromPseudomonas aeruginosaas a highly-efficient genome editing tool forP. putidaandP. aeruginosa. This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second SEVA-vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome ofP. putidaand performed several genomic deletions inP. aeruginosawithin four days, with an efficiency of >83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool forPseudomonas, to complement the existing range of Cas9-based editing methods and facilitates genomic engineering efforts of this important genus.

Importance

The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations inP. putidaandP. aeruginosain a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established SEVA vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets theoriT, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally-occurring plasmids from bacteria.
PAS domain of flagellar histidine kinase FlrB exhibits novel architecture, and binds Heme as sensory signal in unconventional fashion
Authors: Mukherjee Peeali , Agarwal Shubhangi , Basu Mallick Sritapa , Dasgupta Jhimli
Published: June 2023

Phosphorylation of the σ54-dependent transcription activator FlrC by the sensor histidine kinase FlrB is essential in flagellar synthesis ofVibrio cholerae. Despite that, the structure, sensory signal, and mechanistic basis of function of FlrB were elusive. Here we report the crystal structure of the sensory PAS domain of FlrB in functional dimeric state that exhibits a novel architecture. Series of biochemical/biophysical experiments unequivocally established heme as sensory ligand that packs hydrophobically in the ligand binding cleft of FlrB-PAS. Intriguingly, ATP binding to the C-terminal ATP binding (CA) domain assists PAS domain to bind heme, vis-à-vis, heme binding to the PAS facilitates ATP binding to CA; suggesting a synergistic mode of heme and ATP binding to FlrB. We propose that such synergistic binding triggers conformational signaling in FlrB, leading to the downstream flagellar gene transcription. Enhanced swimming motility ofV. choleraewith increased heme uptake further supports this proposition.
The Cognitive Critical Brain: Modulation of Criticality in Task-Engaged Regions
Authors: Liu Xingyu , Fei Xiaotian , Liu Jia
Published: June 2023

The constantly evolving world necessitates a brain that can adapt and respond to rapid changes. The brain, conceptualized as a system performing cognitive functions through collective neural activity, has been shown to maintain a resting state characterized by near-critical neural activity, poised to respond to external stimuli. The dynamic adaptation of nearcriticality during various tasks, however, remains poorly understood. In this study, we utilized the prototypical Hamiltonian Ising model to investigate the modulation of near-criticality in neural activity at the cortical subsystem level during cognitive tasks. Specifically, we theoretically simulated cortical 2D-Ising modelsin silicousing structural MRI data and empirically estimated the system statein vivousing functional MRI data. First, our findings corroborated previous studies that the resting state is typically near-critical as captured by the Ising model. Notably, we found that cortical subsystems changed their criticality levels heterogeneously during a naturalistic movie-watching task, where visual and auditory cortical regions were fine-tuned closer to criticality. A more fine-grained analysis of the ventral temporal cortex during an object recognition task revealed that only regions selectively responsive to a specific object category were tuned closer to criticality when processing that object category. In conclusion, our study supports thecognitive critical brain hypothesisthat modulating the criticality of subsystems within the hierarchical modular brain may be a general mechanism for achieving diverse cognitive functions.
Five ways to waste food: food wasting behaviours questionnaire
Authors: Misiak Michal , Sobol Malgorzata , Sakowski Lukasz , Kowal Marta , Jurczyk Aleksandra , Wojtycka Lidia
Published: June 2023

Purpose

The goal of the present research was to resolve two problems with contemporary methods used to assess consumer food waste: the lack of established categories of food wasting behaviours and difficulties in assessing food waste. In Studies 1 A and 1 B, a five-factor questionnaire for measuring food wasting behaviours was developed. Study 2 and Exploratory analyses verified whether the questionnaire allows for predicting the amount of wasted meat, dairy and bakery and a range of socioeconomic characteristics.

Design/methodology/approach

Based on pre-registered studies, a new questionnaire for measuring the frequency of food wasting behaviours, the Food Wasting Behaviours Questionnaire (FWBQ), was developed.

Findings

The results provided evidence that behaviours associated with food wasting could be narrowed down to five distinctive basic categories: (1) discarding food because of its' unpalatability; (2) preventing food waste through buying only the necessities; (3) preventing food waste through planning; (4) preventing food waste through sharing and (5) preventing food waste through feeding animals. The FWBQ allowed for investigating the socio-economic factors that influence food wasting behaviour. Finally, the FWBQ allowed for predicting the amount of wasted meat, dairy and bakery products. Also, particular factors were associated with a range of socioeconomic characteristics.

Originality/value

The FWBQ has been shown to be an inexpensive and easy-to-use method for systematising distinct categories of food wasting behaviours and demonstrating their determinants. The study takes an empirical approach (rather than intuitive) to distinguish separate categories of food wasting.