bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolic plasticity of T-cell therapies: multi-omic profiling of interacting human tumor-infiltrating lymphocytes and autologous tumor adoptive cell therapy
Authors: Martinez-Paniagua Melisa , Haymaker Cara , Robinson Jonathan , Harel Michal , Creasy Caitlin , Adolacion Jay R T. , An Xingyue , Fathi Mohsen , Rezvan Ali , Kumar Monish , Amritkar Amit , Woodman Scott E. , Amaria Rodabe N. , Geiger Tamar , Hwu Patrick , Bernatchez Chantale , Varadarajan Navin 
Published: September 2022

ABSTRACTAdoptive cell therapy (ACT) based onex vivoexpanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable antitumor responses even in heavily pretreated patients. However, only a subset of patients responds to ACT; efforts to identify correlates of response have focused on profiling the tumor or the TIL but rarely in an interactive environment. Interactive profiling can provide unique insights into the clinical performance of TILs since the fate, function, and metabolism of TILs are influenced by autologous tumor-derived factors. Here, we performed a suite of cell-sparing assays dubbed holistic analysis of the bioactivity of interacting T cells and autologous tumor cells (HABITAT). HABITAT profiling of TILs used for human ACT and their autologous tumor cells included function-based single-cell profiling by timelapse imaging microscopy in nanowell grids (TIMING); multi-omics using RNA-sequencing and proteomics; metabolite inference using genome-scale metabolic modeling, and pulse-chase assays based on confocal microscopy to profile the uptake and fate of fatty acids (FA). Phenotypically, the ACT TILs from both responders (Rs) and nonresponders (NRs) were comprised of predominantly effector memory T cells (TEMcells) and did not express a high frequency of programmed death ligand-1 (PD-L1) and showed no differences in TCR diversity. Our results demonstrate that while tumor cells from both Rs and NRs are efficient at uptaking FAs, R TILs are significantly more efficient at utilizing FA through fatty acid oxidation (FAO) than NR TILs under nutrient starvation conditions. While it is likely that lipid and FA uptake is an inherent adaptation of TIL populations to lipid-rich environments, performing FAO sustains the survival of TILs and allows them to sustain antitumor cytolytic activity. We propose that metabolic plasticity enabling FAO is a desirable attribute of human TILs for ACT leading to clinical responses.
Digital Light Processing 3D printing for biological applications of polydimethylsiloxane-based microfluidics
Authors: Poskus Matthew D. , Wang Tuo , Deng Yuxuan , Borcherding Sydney , Atkinson Jake , Zervantonakis Ioannis K. 
Published: September 2022

AbstractSoft lithography microfluidics offer many benefits over conventional biological assays; however, the impact this field is inhibited by the lack of widespread adoption of this technology in part due to prohibitive cost and fabrication time. Recent improvements in three-dimensional (3D) printing technologies such as digital light processing (DLP) printing offer a cost-effective and rapid prototyping solution to microfluidic fabrication. Limited information is available about how 3D printing parameters and resin cytocompatibility impact the performance of 3D printed molds for fabrication of polydimethylsiloxane (PDMS)-based microfluidics for cellular studies. Using a low-cost, commercially available DLP 3D printer, we assess the cytocompatibility of several resins, optimize printer settings and characterize minimum feature size of our system. We demonstrate the applications of DLP printing for soft lithography microfluidics by developing four assays to characterize cell viability, drug response, establish concentration gradients, and monitor live-cell 3D invasion into a hydrogel.
Subpopulation Codes Permit Information Modulation Across Cortical States
Authors: Getz Matthew P. , Huang Chengcheng , Doiron Brent 
Published: September 2022

AbstractCortical state is modulated by myriad cognitive and physiological mechanisms. Yet it is still unclear how changes in cortical state relate to changes in neuronal processing. Previous studies have reported state dependent changes in response gain or population-wide shared variability, motivated by the fact that both are important determinants of the performance of any population code. However, if the state-conditioned cortical regime is well-captured by a linear input-output response (as is often the case), then the linear Fisher information (FI) about a stimulus available to a decoder is invariant to state changes. In this study we show that by contrast, when one restricts a decoder to a subset of a cortical population, information within the subpopulation can increase through a modulation of cortical state. A clear example of such a subpopulation code is one in which decoders only receive projections from excitatory cells in a recurrent excitatory/inhibitory (E/I) network. We demonstrate the counterintuitive fact that when decoding only from E cells, it is exclusively the I cell response gain and connectivity which govern how information changes. Additionally, we propose a parametrically simplified approach to studying the effect of state change on subpopulation codes. Our results reveal the importance of inhibitory circuitry in modulating information flow in recurrent cortical networks, and establish a framework in which to develop deeper mechanistic insight into the impact of cortical state changes on information processing in these circuits.
Subpopulations of cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer are a predictor of poor clinical outcome
Authors: Mathieson Layla , Koppensteiner Lilian , Pattle Samuel , Dorward David A , O’Connor Richard , Akram Ahsan 
Published: September 2022

AbstractCancer-associated fibroblasts (CAFs) are the dominant cell type in the stroma of solid organ cancers, including non-small cell lung cancer (NSCLC). Fibroblast heterogeneity is widely recognised in many cancers, with subpopulations of CAFs being identified and potentially being indicative of prognosis and treatment efficacy. Here, the subtypes displayed by CAFs isolated from human NSCLC resections are initially identified by flow cytometry, using the markers FAP, CD29, αSMA, PDPN, CD90, FSP-1 and PDGFRβ, showing five distinct subpopulations, CAF-S1-S5. Our findings show that when comparing fibroblasts from tumour tissue with that from adjacent lung tissue, CAF-S2 and CAF-S3 are found in the normal tissue and marker expression suggests a less activated phenotype whereas CAF-S1, CAF-S4 and CAF-S5 are predominantly found in the tumour tissue and are positive for a combination of markers of fibroblast activation. We focus on these subtypes most associated with fibroblast activation, primarily focussing on a previously unreported CAF-S5 subtype, and comparing to the previously identified CAF-S1. Both these subsets express FAP and PDPN as markers of fibroblast activation, but CAF-S5 lacks expression of the common activation marker αSMA. The spatial relevance of these subtypes in a cohort of 163 NSCLC patients was then investigated by multiplex immunofluorescence on a tumour micro-array of patient samples, revealing CAF-S5 are found further from tumour regions than CAF-S1. To understand the functional role of CAF-S5, scRNA sequencing data was used to compare the subset to the previously identified CAF-S1, finding that CAF-S5 displays an inflammatory phenotype, whereas CAF-S1 displays a contractile phenotype. We demonstrate that presence of either the CAF-S1 or CAF-S5 subtype is correlated to worse survival outcome in NSCLC, highlighting the importance of the identification of CAF subtypes in NSCLC.
Over Two Million Protein Translocations Reveal Optimal Conditions for High Bandwidth Recordings, Studying Transport Energetics, and Unfolding
Authors: Nuwan Y. M. , Bandara D. Y. , Freedman Kevin J. 
Published: September 2022

AbstractThe gradual tapered geometry of nanopipettes offers a unique perspective on protein transport through nanopores since both a gradual and fast confinement is possible depending on translocation direction. Protein capture rate, unfolding, speed of translocation, and clogging probability are studied by toggling the lithium chloride concentration between 4 M and 2 M. Interestingly, the proteins in this study could be transported with or against the electrophoresis and offer vastly different attributes of sensing and affect whether a protein unfolds during pore transit. A ruleset for studying proteins is developed that prevents irreversible pore clogging and yielded upwards of >100,000 events/nanopore. Minimizing clogging also permitted higher quality data via the use of smaller pores (i.e., <2× the size of the protein) including higher SNR recordings and data acquisition at the highest available bandwidth (100 kHz). The extended duration of experiments further revealed that the capture rate takes ~2 hours to reach a steady state with a value ~3x greater than the initial reading, emphasizing the importance of reaching equilibrated transport for studying the energetics of protein transport (i.e., diffusion vs barrierlimited). Even in the equilibrated transport state, improper lowpass filtering was shown to distort the classification of diffusion-limited vs barrier-limited transport. Finally electric-field induced protein unfolding was found to be most prominent in EO dominant transport whereas EP dominant events show no evidence of unfolding. Thus, our findings showcase the optimal conditions for protein translocations and the impact on studying protein unfolding, transport energetics, and acquiring high bandwidth data.
CLPB3 is required for the removal of chloroplast protein aggregates and for thermotolerance in Chlamydomonas
Authors: Kreis Elena , Niemeyer Justus , Merz Marco , Scheuring David , Schroda Michael 
Published: September 2022

AbstractIn the cytosol of plant cells, heat-induced protein aggregates are resolved by ClpB/Hsp100 family member HSP101, which is essential for thermotolerance. For chloroplast family member CLPB3 this is less clear with controversial reports on its role in conferring thermotolerance. To shed light onto this issue, we have characterized two Chlamydomonas reinhardtii clpb3 mutants. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TIG1 and the small heat shock proteins HSP22E/F in vivo and for conferring thermotolerance under heat stress. Although CLPB3 accumulates to similarly high levels as stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the ribosomal subunit PRPL1 and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that reduction in chloroplast protein synthesis capacity and increase in protease capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to signals from HSP22E/F, originating largely to the thylakoid membrane occupied area. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.HighlightChloroplast CLPB3 in Chlamydomonas is required for resolving heat-induced protein aggregates and this activity confers thermotolerance under severe heat stress.During heat stress, CLPB3 organizes into stromal foci located next to the thylakoid membrane system, indicating a role for CLPB3 in disentangling protein aggregates from there.
Expanded diversity of tfdA harboring bacteria across the natural and built environment
Authors: White Amber M. , Gonzalez Vazquez Amarilys , McDaniel Elizabeth A. , Peterson Benjamin D. , Koch Paul , Remucal Christina K. , McMahon Katherine D. 
Published: September 2022

Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide commonly used in aquatic and terrestrial environments that is degraded by bacteria through the TFD pathway. Previous work has relied on culture-based methods to develop primers for qPCR analysis of the gene cassette in environmental samples. In this study, we combined molecular and genomic approaches to examine the accuracy of established tfdA qPCR primers on environmental samples and update the phylogeny of tfdA genes detected in bacterial genomes. We found most putative 2,4-D degraders are within the Proteobacteria but also found several novel degraders including members of the phyla Candidatus Rokubacteria and Candidatus Eremiobacteraeota. In silico analysis of established primers showed potential amplification of < 5% of putative degrader sequences but 52-100% of experimentally verified degraders when allowing for three and one mismatches between template and primer sequences, respectively. Overall, our work expands the diversity of putative 2,4-D degraders and demonstrates the limitations of culture-based tools for investigating functional diversity of microorganisms in the environment.ImportanceCultivation-based methods can misrepresent the diversity of environmental microorganisms. Our work showcases one example of how culture-based development of molecular tools underestimates the full spectrum of 2,4-D degrading microorganisms. Accurately identifying microorganisms with 2,4-D degradation potential is crucial for understanding the biodegradation potential of a commonly used herbicide across terrestrial, aquatic, and subsurface environments. Additionally, this work reinforces well-documented pitfalls associated with relying on cultured representatives when constructing primers and the challenges of translating findings from a few cultured representatives to understudied or unknown microorganisms in complex environments.
Hematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic hemogenic endothelium
Authors: Ghersi Joey J. , Baldissera Gabriel , Hintzen Jared , Luff Stephanie A. , Cheng Siyuan , Xia Ivan Fan , Sturgeon Christopher M. , Nicoli Stefania 
Published: September 2022

AbstractMultipotent hematopoietic stem/progenitor cells (HSPCs) generate all mature blood cells in the erythroid, lymphoid, and myeloid lineages. HSPCs are initially produced in the embryo, via transdifferentiation of hemogenic endothelial cells (hemECs) in the aorta-gonad mesonephros (AGM). HSPCs in the AGM are functionally heterogenous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. This knowledge could inform approaches to overcome the dysregulation of HSPC heterogeneity associated with poor outcomes of autologous transplants. Here we discovered that loss of microRNA (miR)-128 (miR-128Δ/Δ) in zebrafish leads to an expansion of hemECs forming replicative HSPCs in the AGM, and a skew towards the erythroid and lymphoid lineages in larval and adult stages. Furthermore, we found that inhibiting miR-128 during the differentiation of human pluripotent stem cells into hemECs, but not during the endothelial-to-hematopoietic transition, recapitulated the lineage skewing. In vivo, expression of wild-type miR-128 in endothelium restored the blood lineage distribution in miR-128Δ/Δ zebrafish. We found that miR-128 represses the expression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b, and thus promotes Wnt and Notch signaling in hemECs. De-repression of cskn1a1 resulted in hemECs generating replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in hemECs forming lymphoid-biased HSPCs in the AGM and relative mature blood cells in adult. We propose that HSPC heterogeneity is established in hemogenic endothelium prior to transdifferentiation and is programmed in part by Wnt and Notch signaling modulation.
Accelerated High-throughput Plant Imaging and Phenotyping System
Authors: Kose Talha , Lins Tiago F. , Wang Jessie , O’Brien Anna M. , Sinton David , Frederickson Megan E. 
Published: September 2022

AbstractThe complex web of interactions in biological communities is an area of study that requires large multifactorial experiments with sufficient statistical power. The use of automated tools can reduce the time and labor associated with experiment setup, data collection, and analysis in experiments aimed at untangling these webs. Here we demonstrate tools for high-throughput experimentation (HTE) in duckweeds, small aquatic plants that are amenable to autonomous experimental preparation and image-based phenotyping. We showcase the abilities of our HTE system in a study with 6,000 experimental units grown across 1,000 different nutrient environments. The use of our automated tools facilitated the collection and analysis of time-resolved growth data, which revealed finer dynamics of plant-microbe interactions across environmental gradients. Altogether, our HTE system can run experiments of up to 11,520 experimental units and can be adapted to studies with other small organisms.
ClickArr: a novel, high throughput assay for evaluating β-arrestin isoform recruitment
Authors: French Alexander R. , Meqbil Yazan J. , van Rijn Richard M. 
Published: September 2022

AbstractModern methods for quantifying signaling bias at GPCRs rely on using a single β-arrestin isoform. However, it is increasingly appreciated that the two β-arrestin isoforms have unique roles, requiring the ability to assess β-arrestin isoform preference. Herein, we present ClickArr, a live-cell assay that simultaneously reports recruitment of both β-arrestin isoforms as they compete for interaction with GPCRs. We demonstrate that an agonist can have β-arrestin isoform bias, potentially opening up a new dimension for drug development.
Balancing competing effects of tissue growth and cytoskeletal regulation duringDrosophilawing disc development
Authors: Kumar Nilay , Tsai Kevin , Mim Mayesha Sahir , Ambriz Jennifer Rangel , Chen Weitao , Zartman Jeremiah J. , Alber Mark 
Published: September 2022

AbstractCytoskeletal structure and force generation within cells must be carefully regulated as the developing organ grows to reach a final size and shape. However, how the complex regulation of multiple features of tissue architecture is simultaneously coordinated remains poorly understood. Through iterations between experiments and novel computational multi-scale model simulations, we investigate the combined regulation of cytoskeletal regulation and proliferation in the growing wing imaginal disc. First, we found through experiments and calibrated model simulations that the local curvature and nuclear positioning of cells in the growing wing disc are defined by patterning of nested spatial domains of peaks in apical and basal contractility. Additionally, predictions from model simulations that incorporate a mechanistic description of interkinetic nuclear migration demonstrate that cell proliferation increases the local basal curvature of the wing disc. This is confirmed experimentally as basal curvature increases when growth and proliferation are increased through insulin signaling. In surprising contrast, we experimentally found that Decapentaplegic (Dpp), the key morphogen involved in both growth control and patterning of the anterior-posterior axis, counteracts increases in tissue bending due to cell proliferation via a combined mechanism that balances the competing impacts of both proliferation and patterning of cell contractility. Overall, the high conservation of these regulatory interactions suggests an important balancing mechanism through dual regulation of proliferation and cytoskeleton to meet the multiple criteria defining tissue morphogenesis.
kcna1a mutant zebrafish as a model of episodic ataxia type 1 and epilepsy
Authors: Dogra Deepika , Meza-Santoscoy Paola L. , Rehak Renata , de la Hoz Cristiane L R , Gavrilovici Cezar , Ibhazehiebo Kingsley , Rho Jong M. , Kurrasch Deborah M. 
Published: September 2022

ABSTRACTObjectiveKCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), with epilepsy as a common comorbidity. Current medications only provide partial relief to ataxia and/or seizures, making new drugs needed. Here, we investigate the utility of zebrafish kcna1a−/− as a model of EA1 with epilepsy by characterizing its phenotype and comparing the efficacy of the first-line therapy carbamazepine in kcna1a−/− zebrafish to Kcna1−/− rodents.MethodsWe used CRISPR/Cas9 mutagenesis to introduce a mutation in the sixth segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a−/− larvae to assess ataxia- and epilepsy-related phenotypes. We also carried out real-time qPCRs to measure the transcript levels of brain hyperexcitability markers and bioenergetic profiling of kcna1a−/− larvae to evaluate their metabolic health. Carbamazepine efficacy was tested using behavioral assessments in kcna1a−/− zebrafish and seizure frequency in Kcna1−/− mice.Resultskcna1a−/− zebrafish showed uncoordinated movements and locomotor deficits. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation. Extracellular field recordings and upregulated fosab transcript levels showed hyperexcitability of the kcna1a−/− brain. Further, vglut2a and gad1b transcript levels were altered, indicative of neuronal excitatory/inhibitory imbalance in the kcna1a−/− brain. Metabolic health was also compromised in kcna1a−/− as seen by a significant reduction in measures of cellular respiration. Notably, carbamazepine reduced the impaired startle response in kcna1a−/− zebrafish but had no effect on the seizure frequency in Kcna1−/− mice, suggesting that this EA1 zebrafish model might better translate to human efficacy compared to rodents.SignificanceWe conclude that zebrafish kcna1a−/− larvae show ataxia and epilepsy-related phenotypes and that they are responsive to carbamazepine treatment, consistent with EA1 patients. This study supports the notion that these zebrafish disease models can be useful for drug screening as well as studying the underlying disease biology.KEY POINTSZebrafish kcna1a−/− larvae display dynamic behavioral changes, along with ataxia-like uncoordinated movements and brain hyperexcitabilitykcna1a−/− larvae have dysfunctional neuronal excitatory/inhibitory balance and perturbed metabolic healthSimilar to its effectiveness in patients, carbamazepine treatment improves behavioral deficits in kcna1a−/− larvae