bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen specific tumor cell killing by cytotoxic T cells
Authors: Mattes Ann-Kathrin , Gamez Marta Puig , Martin Eva , Boryn Lukasz , Baskaran Praveen , Huber Heinrich J. , Schuler Michael , Park John E. , Swee Lee Kim 
Published: August 2022

AbstractCytotoxic CD8+ T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function. CRISPRa and CRISPR KO screens uncovered 186 and 704 hits respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered new genes implicated in tumor resistance to killing. Notably, we discovered thatILKAPencoding the integrin-linked kinase-associated serine/threonine phosphatase 2C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.
Productive and latent HIV infections originate in resting CD4+T cells
Authors: Wietgrefe Stephen W. , Anderson Jodi , Duan Lijie , Southern Peter J. , Zuck Paul , Wu Guoxin , Howell Bonnie J. , Reilly Cavan , Kroon Eugène , Chottanapund Suthat , Buranapraditkun Supranee , Sacdalan Carlo , Tulmethakaan Nicha , Colby Donn J. , Chomchey Nitiya , Prueksakaew Peeriya , Pinyakorn Suteeraporn , Trichavaroj Rapee , Hsu Denise , Vasan Sandhya , Manasnayakorn Sopark , de Souza Mark , Tovanabutra Sodsai , Schuetz Alexandra , Robb Merlin L. , Phanuphak Nittaya , Ananworanich Jintanat , Schacker Timothy W. , Haase Ashley T. 
Published: August 2022

SummaryProductively and latently HIV-infected cells are the source of virus that respectively establishes and sustains systemic infections and the reservoir in which HIV persists and rebounds when anti-retroviral therapy (ART) is interrupted. While infected activated CD4+T cells are thought to be the principal source of HIV production, and reversion of activated infected cells to a resting state as the major pathway to establishment of the latently infected cell reservoir, we now show that in the earliest stages of detectable HIV infection in the lymphoid tissue reservoir, infection of resting CD4+T cells establishes the first populations of both productively and latently infected cells. We further show that the early infection of resting T cells reflects their predominance in lymphoid tissues and the expression of pTEFb in vivo in resting T cells to support their infection. The immediate establishment of productively and latently infected cell populations enable HIV to propagate and persist, and generates reservoirs from which infection can rebound despite instituting ART at the earliest stage of detectable infection.
hERG1 channel subunit composition mediates proton inhibition of IKr in hiPSC-CMs
Authors: Ukachukwu Chiamaka U. , Jimenez-Vazquez Eric N. , Jain Abhilasha , Jones David K. 
Published: August 2022

ABSTRACThERG1 conducts cardiac IKr and is critical for repolarization of the human heart. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden cardiac death. At least two subunits combine to form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG 1a/1b subunit abundance modulates IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue have suggested that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven hiPSC-CM maturation model to investigate how changes in relative hERG 1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immature hiPSC-CMs display a two-fold greater inhibition by extracellular acidosis (pH 6.3) compared to matured hiPSC-CMs. qRT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated, and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared to immature hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by an increased in IKr density. Silencing the impact of hERG1b on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a PAS domain reduced the magnitude of IKr proton inhibition in immature hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and that hERG1 subunit abundance determines IKr sensitivity to protons in hiPSC-CMs.
Ladder shaped microfluidic system enabling rapid antibiotic susceptibility testing with standardized concentration panel
Authors: Nguyen Ann V. , Yaghoobi Mohammad , Azizi Morteza , Davaritouchaee Maryam , Abbaspourrad Alireza 
Published: August 2022

AbstractAntibiotics are often prescribed before pathogens are identified and susceptibility to the prescribed drug is confirmed; laboratory results may take up to 3 days. Using rapid antibiotic susceptibility testing (AST) this timeline can be compressed. We designed a microfluidic ladder-based system that generates a twofold serial dilution of antibiotics comparable to current national and international standards. This consolidated design, with minimal handling steps, has cut down the time-to-result for AST from 16-20 h to 4-5 h. Our system has a 91.75 % rate of agreement with the commercial AST system for Gram-negative and Gram-positive bacterial isolates from canine urinary tract infections (UTI) tested against seven clinically relevant antibiotics. Overall, the system showed a matching rate of 92.71 % - 94.54 % with Gram-negative pathogens, and 85.00 % - 88.57 % with Gram-positive pathogens with no statistical difference between the pathogens or antibiotics. We also tested bacteria filtered directly from urine samples, potentially reducing the total sample-to-result time from 2-3 days to 4 hours.
Overlapping local and systemic defense induced by an oomycete fatty acid MAMP and brown seaweed extract in tomato
Authors: Lewis Domonique C. , Stevens Danielle M. , Little Holly , Coaker Gitta L. , Bostock Richard M. 
Published: August 2022

AbstractEicosapolyenoic fatty acids are integral components of oomycete pathogens that can act as microbe-associated molecular patterns (MAMPs) to induce disease resistance in plants. Defense inducing eicosapolyenoic fatty acids include arachidonic (AA) and eicosapentaenoic acids, and are strong elicitors in solanaceous plants with bioactivity in other plant families. Similarly, extracts of the brown seaweed, Ascophyllum nodosum, used in sustainable agriculture as a biostimulant of plant growth, may also induce disease resistance. A. nodosum, similar to other macroalgae, is rich in eicosapolyenoic fatty acids, which comprise as much as 25% of total fatty acid composition. We investigated the response of roots and leaves from AA or a commercial A. nodosum extract (ANE) on root-treated tomatoes via RNA sequencing, phytohormone profiling, and disease assays. AA and ANE significantly altered transcriptional profiles relative to control plants, inducing numerous defense-related genes with both substantial overlap as well as differences in gene expression patterns. Root treatment with AA and, to a lesser extent, ANE also altered both salicylic acid and jasmonic acid levels while inducing local and systemic resistance to oomycete and bacterial pathogen challenge. Thus, our study highlights overlap in both local and systemic defense induced by AA and ANE, with potential for inducing broad-spectrum resistance against pathogens.
Ferroportin Depletes Iron Needed for Cell Cycle Progression in Head and Neck Squamous Cell Carcinoma
Authors: Belvin B. Ross , Lewis Janina P. 
Published: August 2022

AbstractFerroportin (FPN), the only identified eukaryotic iron efflux channel, plays an important role in iron homeostasis and is down regulated in many cancers. To determine if iron related pathways are important for HNSCC progression and proliferation, we utilize a model of FPN over-expression to simulate iron depletion and probe associated molecular pathways. HNSCC cells are sensitive to iron chelation and ferroptosis, but a non-transformed normal oral keratinocyte (NOK) cell line is not. We found that FPN expression inhibits HNSCC cell proliferation and colony formation but NOK cells are unaffected. Inhibition of cell proliferation is rescued by the addition hepcidin. Decreases in proliferation are due to the disruption of iron homeostasis via loss of labile iron caused by elevated FPN levels. This in turn protects HNSCC cells from ferroptotic cell death. Expression of FPN induces DNA damage, activates p21 and reduces mRNA levels of cyclin proteins thereby inhibiting cell cycle progression of HNSCC cells, arresting cells in S-phase. Induction of FPN severely inhibits Edu incorporation and increases β-galactosidase activity, indicating cells have entered senescence. Finally, in an oral orthotopic mouse xenograft model, FPN induction yields a decrease of tumor growth. Our results indicate that iron plays a role in HNSCC cell proliferation and sustained growth and ferroptosis iron based therapeutic strategies may have potential therapeutic benefit.
Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition
Authors: Gerrick Elias R. , Zlitni Soumaya , West Patrick T. , Carter Matthew M. , Mechler Claire M. , Olm Matthew R. , Caffrey Elisa B. , Li Jessica A. , Higginbottom Steven K. , Severyn Christopher J. , Kracke Frauke , Spormann Alfred M. , Sonnenburg Justin L. , Bhatt Ami S. , Howitt Michael R. 
Published: August 2022

SummaryThe microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we identified several new rodent- and human-associated parabasalid protists. Genomic and metabolomic analyses of murine parabasalids from the genusTritrichomonasrevealed species-level differences in the excretion of the metabolite succinate. This metabolic dissimilarity results in distinct small intestinal immune responses during protist colonization. Metabolic differences betweenTritrichomonasspecies also determine their ecological niche within the microbiota. By manipulating dietary fibers and developingin vitroprotist culture, we show that different parabasalid species preferentially rely on dietary polysaccharides or mucus glycans. These polysaccharide preferences create trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.
Reconstruction of Small Subunit Ribosomal RNA from High-Throughput Sequencing Data: A Comparative Study of Metagenomics and Total RNA Sequencing
Authors: Hempel Christopher A. , Carson Shea E. E. , Elliott Tyler A. , Adamowicz Sarah J. , Steinke Dirk 
Published: August 2022

AbstractThe small subunit (SSU) ribosomal RNA (rRNA) is the most commonly used marker for the identification of microbial taxa, but its full-length reconstruction from high-throughput sequencing (HTS) data remains challenging, especially for complex and diverse environmental samples. Metagenomics and total RNA sequencing (total RNA-Seq) are target-PCR-free HTS methods that are used to characterize microbial communities and simultaneously reconstruct SSU rRNA sequences. However, more testing is required to determine and improve their effectiveness. In this study, we processed metagenomics and total RNA-Seq data retrieved from a commercially available mock microbial community using 112 combinations of commonly used data-processing tools, determined SSU rRNA reconstruction completeness of both sequencing methods for each species in the mock community, and analyzed the impact of data-processing tools on SSU rRNA and genome completeness. Total RNA-Seq allowed for the complete or near-complete reconstruction of all mock community SSU rRNA sequences and outperformed metagenomics. SSU rRNA completeness of metagenomics strongly correlated with the genome size of mock community species. The impact of data-processing tools was overall low, although certain tools resulted in significantly lower SSU rRNA completeness. These results are promising for the high-throughput reconstruction of novel full-length SSU rRNA sequences and could advance the simultaneous application of multiple -omics approaches in routine environmental assessments to allow for more holistic assessments of ecosystems.
Large-scale mapping and systematic mutagenesis of human transcriptional effector domains
Authors: DelRosso Nicole , Tycko Josh , Suzuki Peter , Andrews Cecelia , Aradhana  , Mukund Adi , Liongson Ivan , Ludwig Connor , Spees Kaitlyn , Fordyce Polly , Bassik Michael C. , Bintu Lacramioara 
Published: August 2022

SummaryHuman gene expression is regulated by over two thousand transcription factors and chromatin regulators1,2. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of transcriptional effector domains they contain, their location in the protein, their activation and repression strengths, and the amino acids that are necessary for their functions. Here, we systematically measure the transcriptional effector activity of >100,000 protein fragments (each 80 amino acids long) tiling across most chromatin regulators and transcription factors in human cells (2,047 proteins). By testing the effect they have when recruited at reporter genes, we annotate 307 new activation domains and 592 new repression domains, a ∼5-fold increase over the number of previously annotated effectors3,4. Complementary rational mutagenesis and deletion scans across all the effector domains reveal aromatic and/or leucine residues interspersed with acidic, proline, serine, and/or glutamine residues are necessary for activation domain activity. Additionally, the majority of repression domain sequences contain either sites for SUMOylation, short interaction motifs for recruiting co-repressors, or are structured binding domains for recruiting other repressive proteins. Surprisingly, we discover bifunctional domains that can both activate and repress and can dynamically split a cell population into high- and low-expression subpopulations. Our systematic annotation and characterization of transcriptional effector domains provides a rich resource for understanding the function of human transcription factors and chromatin regulators, engineering compact tools for controlling gene expression, and refining predictive computational models of effector domain function.
Multiplex editing of theNucleoredoxin1tandem array in poplar: from small indels to translocations and complex inversions
Authors: Chen Yen-Ho , Sharma Shakuntala , Bewg William P. , Xue Liang-Jiao , Gizelbach Cole R. , Tsai Chung-Jui 
Published: August 2022

AbstractThe CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. Here, we used CRISPR-Cas9 to mutagenize a tandem array of sevenNucleoredoxin1 (NRX1) genes spanning ~100 kb inPopulus tremula×alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid functional characterization.
The archaeal KEOPS complex possesses a functional Gon7 homolog and has an essential function independent of cellular t6A modification level
Authors: Wu Pengju , Gan Qi , Zhang Xuemei , Yang Yunfeng , Xiao Yuanxi , She Qunxin , Ni Jinfeng , Huang Qihong , Shen Yulong 
Published: August 2022

AbstractKEOPS is a multi-subunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N6-threonylcarbamoyl adenosine (t6A) modification of tRNAs. Recently, KEOPS is reported to participate in homologous recombination repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeonSaccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1-like (or Gon7-like), just as eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t6A modification complementary strain named TsaKI, implying that aKEOPS complex is essential for an additional cellular process in this archaeon. Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type which is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of cellular t6A modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity which relies on its tRNA 3’ CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests of an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t6A modification and the additional function presumably homologous recombination.
Inoculum and disease dynamics of citrus greasy spot caused by Zasmidium citri-griseum in sweet orange in Panama
Authors: Aguilera-Cogley Vidal , Sedano Elena , Vicent Antonio 
Published: August 2022

AbstractCitrus greasy spot, caused by Zasmidium citri-griseum, is a disease characterized by inducing premature defoliation and a reduction in yield in different citrus species. Greasy spot is the most prevalent fungal disease in sweet orange in Panama. Nevertheless, no epidemiological information is available. In this study, the dynamics of the defoliation, inoculum production, airborne inoculum, and infection periods of Z. citri-griseum and their associations with environmental conditions were determined in Panama. The period from December to April was characterized by greater defoliation of trees, with the greatest amount of leaf litter being produced in January and February. The number of days until total leaf decomposition (DLD) was related to the number of rainy days >1mm (NRD), accumulated rainfall (AR), and average relative humidity (ARH). The number of ascospores released from leaf litter (ASCL) was related to DLD, NRD, AR, and average temperature (AT). The greatest amounts of airborne ascospores (AASC) of Z. citri-griseum occur during April and May, when the rainy season begins in Panama. Similarly, the highest incidence (INC) of greasy spot in the trap plants coincided with the months of the greatest availability of airborne ascospores. However, infections were also recorded during other times of the year. The AASC or INC data were fitted to Bayesian models including meteorological variables and an autoregressive temporal component, the latter being the most influential. The results obtained in this study will allow the development of more efficient and sustainable fungicide programs for greasy spot control in Panama.
Engineered nickel bioaccumulation in Escherichia coli by NikABCDE transporter and metallothionein overexpression
Authors: Diep P. , Shen H. , Wiesner J. A. , Mykytczuk N. , Papangelakis V. , Yakunin A. F. , Mahadevan R. 
Published: August 2022

ABSTRACTMine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7-fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.