bluuebunny/crossref_metadata_2025 huggingface dataset:
Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5
Authors: McComas Sarah E. , Reichenbach Tom , Mitrovic Darko , Alleva Claudia , Bonaccorsi Marta , Delemotte Lucie , Drew David
Published: June 2022

In mammals, glucose transporters (GLUT) control organism-wide blood glucose homeostasis. In human, this is accomplished by fourteen different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarialplasmodium falciparumtransporterPfHT1, which is uniquely able to transport a wide range of different sugars.PfHT1 was captured in an intermediate “occluded” state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity inPfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed inPfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resemblesPfHT1. The coordination of fructose lowers the energetic barriers between the outward and inward-facing states, and the observed binding mode for fructose is consistent with biochemical analysis. Rather than a substrate binding site that achieves strict specificity by having a high-affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.
Genetic regulation of the human plasma proteome in 54,306 UK Biobank participants
Authors: Sun Benjamin B. , Chiou Joshua , Traylor Matthew , Benner Christian , Hsu Yi-Hsiang , Richardson Tom G. , Surendran Praveen , Mahajan Anubha , Robins Chloe , Vasquez-Grinnell Steven G. , Hou Liping , Kvikstad Erika M. , Burren Oliver S. , Cule Madeleine , Davitte Jonathan , Ferber Kyle L. , Gillies Christopher E. , Hedman Åsa K. , Hu Sile , Lin Tinchi , Mikkilineni Rajesh , Pendergrass Rion K. , Pickering Corran , Prins Bram , Raj Anil , Robinson Jamie , Sethi Anurag , Ward Lucas D. , Welsh Samantha , Willis Carissa M. ,   Alnylam Human Genetics,   AstraZeneca Genomics Initiative,   Biogen Biobank Team,   Bristol Myers Squibb,   Genentech Human Genetics,   GlaxoSmithKline Genomic Sciences,   Pfizer Integrative Biology,   Population Analytics of Janssen Data Sciences,   Regeneron Genetics Center, Burkitt-Gray Lucy , Black Mary Helen , Fauman Eric B. , Howson Joanna M. M. , Kang Hyun Min , McCarthy Mark I. , Melamud Eugene , Nioi Paul , Petrovski Slavé , Scott Robert A. , Smith Erin N. , Szalma Sándor , Waterworth Dawn M. , Mitnaul Lyndon J. , Szustakowski Joseph D. , Gibson Bradford W. , Miller Melissa R. , Whelan Christopher D.
Published: June 2022

The UK Biobank Pharma Proteomics Project (UKB-PPP) is a collaboration between the UK Biobank (UKB) and thirteen biopharmaceutical companies characterising the plasma proteomic profiles of 54,306 UKB participants. Here, we describe results from the first phase of UKB-PPP, including protein quantitative trait loci (pQTL) mapping of 1,463 proteins that identifies 10,248 primary genetic associations, of which 85% are newly discovered. We also identify independent secondary associations in 92% of cis and 29% of trans loci, expanding the catalogue of genetic instruments for downstream analyses. The study provides an updated characterisation of the genetic architecture of the plasma proteome, leveraging population-scale proteomics to provide novel, extensive insights into trans pQTLs across multiple biological domains. We highlight genetic influences on ligand-receptor interactions and pathway perturbations across a diverse collection of cytokines and complement proteins, and illustrate long-range epistatic effects of ABO blood group and FUT2 secretor status on proteins with gastrointestinal tissue-enriched expression. We demonstrate the utility of these data for drug target discovery by extending the genetic proxied effect of PCSK9 levels on lipid concentrations, cardio- and cerebro-vascular diseases, and additionally disentangle specific genes and proteins perturbed at COVID-19 susceptibility loci. This public-private partnership provides the scientific community with an open-access proteomics resource of unprecedented breadth and depth to help elucidate biological mechanisms underlying genetic discoveries and accelerate the development of novel biomarkers and therapeutics.
FunSpace: A functional and spatial analytic approach to cell imaging data using entropy measures
Authors: Vu Thao , Seal Souvik , Wrobel Julia , Ghosh Tusharkanti , Ahmadian Mansooreh , Ghosh Debashis
Published: June 2022

Spatial heterogeneity in the tumor microenvironment (TME) plays a critical role in gaining insights into tumor development and progression. Conventional metrics typically capture the spatial differential between TME cellular architectures by either exploring the cell distributions in a pairwise fashion or aggregating the heterogeneity across multiple cell distributions without considering for the spatial contribution. As such, none of the existing approaches has fully accounted for the heterogeneity caused by both cellular diversity and spatial configurations of multiple cell categories. In this article, we propose an approach to leverage the spatial entropy measures at multiple distance ranges to account for the spatial heterogeneity across different cellular architectures. Then, functional principal component analysis (FPCA) targeting sparse data is applied to estimate FPC scores which are then predictors in a Cox regression model to investigate the impact of spatial heterogeneity in the TME on survival outcome, holding other clinical variables constant. Using an ovarian cancer dataset (n = 114) as a case study, we found that the spatial heterogeneity in the TME immune compositions of CD19+ B cells, CD4+ T cells, CD8+ T cells, and CD68+ macrophages, had a significant non-zero effect on the overall survival (p = 0.027). In the simulations studies under different spatial configurations, the proposed method demonstrated a high predictive power by accounting for both clinical effect and the impact of spatial heterogeneity.
TheLegionella-driven PtdIns(4)Pgradient at LCV-ER membrane contact sites promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole remodeling
Authors: Vormittag Simone , Hüsler Dario , Haneburger Ina , Kroniger Tobias , Anand Aby , Prantl Manuel , Barisch Caroline , Maaß Sandra , Becher Dörte , Letourneur François , Hilbi Hubert
Published: June 2022

The causative agent of Legionnaires’ disease,Legionella pneumophila, governs interactions with host cells by secreting ca. 330 different “effector” proteins. The facultative intracellular bacteria replicate in macrophages and amoeba within a unique compartment, theLegionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide (PI) lipid conversion from PtdIns(3)Pto PtdIns(4)P, fusion with endoplasmic reticulum (ER)-derived vesicles and a tight association with the ER. Proteomics of purified LCVs revealed the presence of membrane contact sites (MCS) proteins implicated in lipid exchange. Using dually fluorescence-labeledDictyostelium discoideumamoeba, we reveal that the VAMP-associated protein (Vap), the PtdIns(4)P4-phosphatase Sac1, and the large fusion GTPase Sey1/atlastin-3 localize to the ER, but not to the LCV membrane, and that these ER-resident proteins promote intracellular replication ofL. pneumophilaand LCV remodeling. Moreover, oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and promote (OSBP8) or restrict (OSBP11) intracellular replication ofL. pneumophilaand LCV expansion. Furthermore, the PtdIns(4)P-subvertingL. pneumophilaeffectors LepB and SidC also promote LCV remodeling. Taken together, theLegionella- and host cell-driven PtdIns(4)Pgradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole remodeling.
Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation
Authors: Schmassmann Philip , Roux Julien , Dettling Steffen , Hogan Sabrina , Shekarian Tala , Martins Tomás A. , Ritz Marie-Françoise , Herter Sylvia , Bacac Marina , Hutter Gregor
Published: June 2022

Glioblastoma (GBM) harbors a highly immunosuppressive tumor microenvironment (TME) which influences glioma growth. Major efforts have been undertaken to describe the TME on a single-cell level. However, human data on regional differences within the TME remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on paired biopsies from the tumor center, peripheral infiltration zone and blood of five primary GBM patients. Through analysis of > 45’000 cells, we revealed a regionally distinct transcription profile of microglia (MG) and monocyte-derived macrophages (MdMs) and an impaired activation signature in the tumor-peripheral cytotoxic-cell compartment. Comparing tumor-infiltrating CD8+T cells with circulating cells identified CX3CR1highand CX3CR1intCD8+T cells with effector and memory phenotype, respectively, enriched in blood but absent in the TME. Tumor CD8+T cells displayed a tissue-resident memory phenotype with dysfunctional features. Our analysis provides a regionally resolved mapping of transcriptional states in GBM-associated leukocytes, serving as an additional asset in the effort towards novel therapeutic strategies to combat this fatal disease.
Comparison of missing data handling methods for variant pathogenicity predictors
Authors: Särkkä Mikko Ilmari , Myöhänen Sami , Marinov Kaloyan , Saarinen Inka , Lahti Leo , Fortino Vittorio , Paananen Jussi
Published: June 2022

Background

Modern clinical genetic tests utilize next-generation sequencing (NGS) approaches to comprehensively analyze genetic variants from patients. Out of these millions of variants, clinically relevant variants that match the patient's phenotype need to be identified accurately within a rapid timeframe that facilitates clinical action. As manual evaluation of variants is not a feasible option for meeting the speed and volume requirements of clinical genetic testing, automated solutions are needed. Various machine learning (ML), artificial intelligence (AI), and in silico variant pathogenicity predictors have been developed to solve this challenge. These solutions rely on the comprehensiveness of the available data and struggle with the sparse nature of genetic variant data. Therefore, careful treatment of missing data is necessary, and the selected methods may have a huge impact on the accuracy, reliability, speed and associated computational costs.

Results

We present an open-source framework called AMISS that can be used to evaluate performance of different methods for handling missing genetic variant data in the context of variant pathogenicity prediction. Using AMISS, we evaluated 14 methods for handling missing values. The performance of these methods varied substantially in terms of precision, computational costs, and other attributes. Overall, simpler imputation methods and specifically mean imputation performed best.

Conclusions

Selection of the missing data handling method is crucial for AI/ML-based classification of genetic variants. We show that utilizing sophisticated imputation methods is not worth the cost when used in the context of genetic variant pathogenicity classification.
The mechanosensitive ion channel Piezo1 modulates the migration and immune response of microglia
Authors: Zhu Ting , Kala Shashwati , Guo Jinghui , Wu Yong , Chen Hui , Zhu Jiejun , Wong Kin Fung , Cheung Chi Pong , Huang Xiaohui , Zhao Xinyi , Lei Ting , Yang Minyi , Sun Lei
Published: June 2022

Microglia are the brain’s resident immune cells, performing surveillance to promote homeostasis and healthy functioning. While chemical signaling in microglia is well-studied, the mechanical cues regulating their function are less well-understood. Here, we investigate the role of the mechanosensitive ion channel Piezo1 in microglia migration, pro-inflammatory cytokine production and stiffness sensing. We demonstrated the functional expression of Piezo1 in microglia and identified genes whose expression was affected by conditional Piezo1 knockout in transgenic mice. Functional assays revealed that Piezo1-deficiency in microglia enhanced migration towards amyloid β-protein, and decreased levels of pro-inflammatory cytokines produced upon stimulation by lipopolysaccharide, both in vitro and in vivo. The phenomenon could be mimicked or reversed using a Piezo1-specific agonist or antagonist. Finally, we also showed that Piezo1 mediated the effect of substrate stiffness-induced migration and cytokine expression. Altogether, we show that Piezo1 is an important molecular mediator for microglia, its activation modulating their migration and immune responses.
Sex-Specific Role of Myostatin Signaling in Neonatal Muscle Growth, Denervation Atrophy, and Neuromuscular Contractures
Authors: Emmert Marianne E , Aggarwal Parul , Shay-Winkler Kritton , Lee Se-Jin , Goh Qingnian , Cornwall Roger
Published: June 2022

Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders.
‘’Reproductive response of laying chickens to ameliorative method of aflatoxin
Authors: Ojo Olayinka Abosede , Bitto Immanuel , Ewuola Emmanuel Olubisi
Published: June 2022

Aflatoxin is toxic, carcinogenic and ubiquitous in nature, affecting both crops and livestock. Mitigating aflatoxin effect using toxin binders has not been very effective. Information on the use of biological methods in aflatoxin mitigation has not been adequately documented. Consequently, influence of bio-control method of aflatoxin on some reproductive hormones, ovarian weight and histopathological parameters of laying chickens (LC) were investigated. Point-of-lay Bovan Nera (n=700) were harphazardly distributed to four dietary treatments; Aflasafe maize-based diet (AMBD), farm feed (FF), aflatoxin-contaminated diet with toxin binder (ACDTB) and aflatoxin-contaminated diet without toxin binder (ACDWTB). The contaminated diets contained 306.3ppb aflatoxin and the experimental design was completely randomized into four treatments (n= 175) of five replicates (n=35) per treatment for a period of 14 weeks. Blood (5mL) was collected at 14th week for LC to determine the estrogen, luteinising hormone (LH), follicle stimulating hormone (FSH), histopathology of the ovary using standard procedures. Data were analysed using descriptive statistics and ANOVA at a0.05. The ROW (%) ranged from 0.34±0.2 (ACDTB) to 0.93±0.3 (AMBD). Estrogen (mg/dL) value was highest in LC fed ACDWTB (2.75±1.08) and least in FF (2.07±0.52). The LH (iu/L) value was highest in LC fed AMBD (1.29±1.68) and least in ACDTB (0.36±0.32). Histopathology of the ovary showed cysts, observed along the oviduct wall in LC fed ACDTB. AMBD enhanced active laying period in LC with no sign of aflatoxocosis. The use of aflasafe maize grain in poultry diet is recommended.
GRINtrode: A neural implant for simultaneous two-photon imaging and extracellular electrophysiology in freely moving animals
Authors: McCullough Connor M. , Ramirez-Gordillo Daniel , Hall Michael , Futia Gregory L. , Moran Andrew K. , Gibson Emily A. , Restrepo Diego
Published: June 2022

Significance

In vivo imaging and electrophysiology are powerful tools to explore neuronal function that each offer unique complementary information with advantages and limitations. Capturing both data types from the same neural population in the freely moving animal would allow researchers to take advantage of the capabilities of both modalities and further understand how they relate to each other.

Aim

Here we present a head-mounted neural implant suitable for in vivo two-photon imaging of neuronal activity with simultaneous extracellular electrical recording in head-fixed or freely moving animals.

Approach

A GRIN lens-based head-mounted neural implant with extracellular electrical recording provided by tetrodes on the periphery of the GRIN lens was chronically implanted. The design of the neural implant allows for recording from head-fixed animals, as well as freely moving animals by coupling the imaging system to a coherent imaging fiber bundle.

Results

We demonstrate simultaneous two-photon imaging of GCaMP and extracellular electrophysiology of neural activity in awake head-fixed, and freely moving mice. Using the collected information, we perform correlation analysis to reveal positive correlation between optical and local field potential recordings.

Conclusion

Simultaneously recording neural activity using both optical and electrical methods provides complementary information from each modality. Designs that can provide such bimodal recording in freely moving animals allow for the investigation of neural activity underlying a broader range of behavioral paradigms.
Comparison of B values obtained by two methodologies that use soil as a substrate and their influence on the estimation of the proportion of nitrogen fixed by legumes
Authors: Berriel Verónica , Monza Jorge , Perdomo Carlos
Published: June 2022

The B value is required to quantify the nitrogen derived from the atmosphere (%Ndfa) in the Rhizobium-legume symbiosis using the 15N natural abundance method. When the B value of a particular specie is not known, one possibility Is to use as a proxy the B value of a specie from the same genus, but this can cause the estimate of %Ndfa to be inaccurate. In this work, we compared two methodologies for determining the B value of Crotalaria juncea, C. spectabilis, C. ochroleuca and Cajanus cajan, using soil as the substrate. One method involvedgrowing plants in soil and averaging the lowest δ15N values of plant shoots (B-minimum), while the other consisted in adding sucrose to soil to immobilize the mineral nitrogen (N-immobilized), and then averaging the shoot δ15N values of all plants. Results showed that B values of C. cajan and C. ochroleuca obtained using the N-immobilized method were up to 1‰ lower than those reported in the literature for these species. Therefore, we propose that, at least in these species, B values determined with the N-immobilized method should be used to estimate the%Ndfa.
Shared enhancer gene regulatory networks between wound and oncogenic programs
Authors: Floc’hlay Swann , Balaji Ramya , Christiaens Valerie , González-Blas Carmen Bravo , De Winter Seppe , Hulselmans Gert , De Waegeneer Maxime , Quan Xiaojiang , Koldere Duygu , Atkins Mardelle , Halder Georg , Classen Anne , Aerts Stein
Published: June 2022

Wound response programs are often activated during neoplastic growth in tumors. In both wound repair and tumor growth, cells respond to acute stress and balance the activation of multiple programs including apoptosis, proliferation, and cell migration. Central to those responses are the activation of the JNK/MAPK and JAK/STAT signaling pathways. Yet, to what extent these signaling cascades interact at the cis-regulatory level, and how they orchestrate different regulatory and phenotypic responses is still unclear. Here, we aim to characterize the regulatory states that emerge and cooperate in the wound response, using the Drosophila melanogaster wing disc as a model system, and compare these with cancer cell states induced by rasV12scrib-/- in the eye disc. We used single-cell multiome profiling to derive enhancer Gene Regulatory Networks (eGRNs) by integrating chromatin accessibility and gene expression signals. We identify a “proliferative” eGRN, active in the majority of wounded cells and controlled by AP-1 and STAT. In a smaller, but distinct population of wound cells, a “senescent” eGRN is activated and driven by C/EBP-like transcription factors (Irbp18, Xrp1, Slow border, and Vrille) and Scalloped. In tumor cells on the other hand, both eGRN signatures are simultaneously active within the same cell. Our single-cell multiome and eGRNs resource offers an in-depth characterisation of the senescence markers, together with a new perspective on the shared gene regulatory programs acting during wound response and oncogenesis.
GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis
Authors: Cordova Ricardo A. , Misra Jagannath , Amin Parth H. , Klunk Angela J. , Damayanti Nur P. , Carlson Kenneth R. , Elmendorf Andrew J. , Kim Hyeong-Geug , Mirek Emily T. , Elzey Bennet D. , Miller Marcus J. , Dong X. Charlie , Cheng Liang , Anthony Tracy G. , Pili Roberto , Wek Ronald C. , Staschke Kirk A.
Published: June 2022

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

Significance

The ISR kinase GCN2 is critical for maintaining tumor amino acid levels to facilitate growth, suggesting a novel therapeutic strategy for the treatment of prostate cancer by inducing starvation for essential amino acids.
IMPPAT 2.0: an enhanced and expanded phytochemical atlas of Indian medicinal plants
Authors: Vivek-Ananth R. P. , Mohanraj Karthikeyan , Sahoo Ajaya Kumar , Samal Areejit
Published: June 2022

Compilation, curation, digitization and exploration of the phytochemical space of Indian medicinal plants can expedite ongoing efforts toward natural product and traditional knowledge based drug discovery. To this end, we present IMPPAT 2.0, an enhanced and expanded database, compiling manually curated information on 4010 Indian medicinal plants, 17967 phytochemicals, 1095 therapeutic uses and 1133 traditional Indian medicinal formulations. Notably, IMPPAT 2.0 compiles associations at the level of plant parts, and provides a FAIR compliant non-redundant in silico stereo-aware library of 17967 phytochemicals from Indian medicinal plants. The phytochemical library has been annotated with several useful properties to enable easier exploration of the chemical space. We also filtered a subset of 1335 drug-like phytochemicals of which majority have no similarity to existing approved drugs. Using cheminformatics, we have characterized the molecular complexity and molecular scaffold based structural diversity of the phytochemical space of Indian medicinal plants, and performed a comparative analysis with other chemical libraries. Altogether, IMPPAT is the largest phytochemical atlas of Indian medicinal plants which is accessible at: https://cb.imsc.res.in/imppat/.
Generating New Musical Preferences from Multi-level Mapping of Predictions to Reward
Authors: Kathios Nicholas , Sachs Matthew E. , Zhang Euan , Ou Yongtian , Loui Psyche
Published: June 2022

Much of what we know and love about music hinges on our ability to make successful predictions, which appears to be an intrinsically rewarding process. Yet the exact process by which learned predictions become pleasurable is unclear. Here, we created novel melodies in an alternative scale different from any established musical culture, to show how musical preference is generatedde novo. Across nine studies (n=1185), participants learned to like more frequently-presented items that adhered to this rapidly-learned structure, suggesting that exposure and prediction errors both affected self-report liking ratings. Learning trajectories varied by music reward sensitivity, but were similar for USA and Chinese participants. Furthermore, fMRI activity in auditory areas reflected prediction errors whereas functional connectivity between auditory and medial prefrontal regions reflected both exposure and prediction errors. Collectively, results support predictive coding as a cognitive mechanism by which new musical sounds become rewarding.
GAVISUNK: Genome assembly validation via inter-SUNK distances in Oxford Nanopore reads
Authors: Dishuck Philip C. , Rozanski Allison N. , Logsdon Glennis A. , Eichler Evan E.
Published: June 2022

Motivation

Highly contiguous de novo genome assemblies are now feasible for large numbers of species and individuals. Methods are needed to validate assembly accuracy and detect misassemblies with orthologous sequencing data to allow for confident downstream analyses.

Results

We developed GAVISUNK, an open-source pipeline that detects misassemblies and produces a set of reliable regions genome-wide by assessing concordance of distances between unique k-mers in Pacific Biosciences high-fidelity (HiFi) assemblies and raw Oxford Nanopore Technologies reads.

Availability

GAVISUNK is available at https://github.com/pdishuck/GAVISUNK.

Contact

eee@gs.washington.edu
Limited role of generation time changes in driving the evolution of mutation spectrum in humans
Authors: Gao Ziyue , Zhang Yulin , Cramer Nathan , Przeworski Molly , Moorjani Priya
Published: June 2022

Recent studies have suggested that the human germline mutation rate and spectrum evolve rapidly. Variation in generation time has been linked to these changes, though its contribution remains unclear. We develop a framework to characterize temporal changes in polymorphisms within and between populations, while controlling for the effects of natural selection and biased gene conversion. Application to the 1000 Genomes Project dataset reveals multiple independent changes that arose after the split of continental groups, including a previously reported, transient elevation in TCC>TTC mutations in Europeans and novel signals of divergence in C>G and T>A mutation rates among population samples. We also find a significant difference between groups sampled in and outside of Africa, in old T>C polymorphisms that predate the out-of-Africa migration. This surprising signal is driven by TpG>CpG mutations, and stems in part from mis-polarized CpG transitions, which are more likely to undergo recurrent mutations. Finally, by relating the mutation spectrum of polymorphisms to parental age effects onde novomutations, we show that plausible changes in the generation time cannot explain the patterns observed for different mutation types jointly. Thus, other factors--genetic modifiers or environmental exposures--must have had a non-negligible impact on the human mutation landscape.
Sparse-spectral microendoscopy for real-time visualization of tumor cell phenotype and microenvironment spatial heterogeneity in vivo
Authors: Spring Bryan Q. , Palanisami Akilan , Saad Mohammad Ahsan , Kercher Eric M. , Lang Ryan T. , Harman Rebecca C. , Sutin Jason , Mai Zhiming , Hasan Tayyaba
Published: June 2022

Cancer heterogeneity and its transformation with time propels treatment resistance and confounds patient outcomes. The inability to monitor in vivo the low abundance, heterocellular phenotypes that resist treatment and ultimately lead to patient death limits the ability to design precision therapies. Here we overcome limitations in multiplexed fluorescence phenotyping to introduce real-time, cellular resolution visualization of tumor heterogeneity in vivo. This method was performed to simultaneously map for the first time 5 individual biomarkers of stemness, proliferation, metabolism, leukocytes and angiogenesis deep within the peritoneal cavities of micrometastatic cancer mouse models at 17 frames per second (fps). The newly developed imaging system revealed distinct cancer cell phenotype–immune cell spatial correlations and clearly visualized the dynamic spatial response of resistant cancer cell niches following treatment. Furthermore, wide-field datasets were generated to facilitate derivation of a mathematical framework for quantifying biomarker spatial variation and thereby overcoming the area restrictions of conventional tumor biopsy. These results pave the way for real-time identification of cancer cell phenotypes in a clinical setting, on which optimized treatment regimens can be based for personalized treatment and precision therapy e.g., tumor margin determination during surgical resection. Additionally, this modality can be used to obtain more fundamental insights into tumor heterogeneity and how treatments affect the molecular and cellular responses of patient-specific disease.
Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells
Authors: Chew Katherine , Lee Branden , van Haren Simon D. , Nanishi Etsuro , O’Meara Timothy , Splaine Jennifer B. , DeLeon Maria , Soni Dheeraj , Seo Hyuk-Soo , Dhe-Paganon Sirano , Ozonoff Al , Smith Jennifer A. , Levy Ofer , Dowling David J.
Published: June 2022

We describe a methodology utilizing high-throughput and high-content screening for novel adjuvant candidates that was used to screen a library of ∼2,500 small molecules via a 384-well quantitative combined cytokine and flow cytometry immunoassay in primary human peripheral blood mononuclear cells (PBMCs) from 4 healthy adult study participants. Hits were identified based on their induction of soluble cytokine (TNF, IFNγ and IL-10) secretion and PBMC maturation (CD 80/86, Ox40, and HLA-DR) in at least two of the four donors screened. From an initial set of 197 compounds identified using these biomarkers—an 8.6% hit rate—we downselected to five scaffolds that demonstrated robust efficacy and potencyin vitroand evaluated the top hit, vinblastine sulfate, for adjuvanticityin vivo. Vinblastine sulfate significantly enhanced murine humoral responses to recombinant SARS-CoV-2 spike protein, including a four-fold enhancement of IgG titer production when compared to treatment with the spike antigen alone. Overall, we outline a methodology for discovering immunomodulators with adjuvant potential via high-throughput screening of PBMCsin vitrothat yielded a lead compound within vivoadjuvanticity.

Motivation

Vaccines are a key biomedical intervention to prevent the spread of infectious diseases, but their efficacy can be limited by insufficient immunogenicity and nonuniform reactogenic profiles. Adjuvants are molecules that potentiate vaccine responses by inducing innate immune activation. However, there are a limited number of adjuvants in approved vaccines, and current approaches for preclinical adjuvant discovery and development are inefficient. To enhance adjuvant identification, we developed a protocol based onin vitroscreening of human primary leukocytes.
Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii
Authors: Dalvie Neil C. , Naranjo Christopher A. , Rodriguez-Aponte Sergio A. , Johnston Ryan S. , Love J. Christopher
Published: June 2022

Background

Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly.

Results

We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein.

Conclusions

Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy will enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.
Compensatory epistasis maintains ACE2 affinity in SARS-CoV-2 Omicron BA.1
Authors: Moulana Alief , Dupic Thomas , Phillips Angela M. , Chang Jeffrey , Nieves Serafina , Roffler Anne A. , Greaney Allison J. , Starr Tyler N. , Bloom Jesse D. , Desai Michael M.
Published: June 2022

The Omicron BA.1 variant emerged in late 2021 and quickly spread across the world. Compared to the ancestral Wuhan Hu-1 strain and other pre-Omicron SARS-CoV-2 variants, BA.1 has many mutations, a number of which are known to enable antibody escape 1–3. Many of these antibody-escape mutations individually decrease the spike receptor-binding domain (RBD) affinity for ACE2 in the background of early SARS-CoV-2 variants 4, but BA.1 still binds ACE2 with high affinity 5,6. The fitness and evolution of the BA.1 lineage is therefore driven by the combined effects of numerous mutations. Here, we systematically map the epistatic interactions between the 15 mutations in the RBD of BA.1 relative to the Wuhan Hu-1 strain. Specifically, we measure the ACE2 affinity of all possible combinations of these 15 mutations (2 15 = 32,768 genotypes), spanning all possible evolutionary intermediates from the ancestral Wuhan Hu-1 strain to BA.1. We find that immune escape mutations in BA.1 individually reduce ACE2 affinity but are compensated by epistatic interactions with other affinity-enhancing mutations, including Q498R and N501Y. Thus, the ability of BA.1 to evade immunity while maintaining ACE2 affinity is contingent on acquiring multiple interacting mutations. Our results implicate compensatory epistasis as a key factor driving substantial evolutionary change for SARS-CoV-2 and are consistent with Omicron BA.1 arising from a chronic infection.
EWSR1 prevents the induction of aneuploidy by regulating the localization of Aurora B at inner centromere
Authors: Kim Haeyoung , Park Hyewon , Schulz Evan T. , Azuma Yoshiaki , Azuma Mizuki
Published: June 2022

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5’-end using CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at kinetochores compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 is essential to prevent aneuploidy through interaction with Aurora B, most likely by regulating the localization of Aurora B at centromere.
Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila
Authors: Ding Kate , Barretto Elizabeth C , Johnston Michael , Lee Byoungchun , Gallo Marco , Grewal Savraj S
Published: June 2022

When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue- and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. We previously identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. Here we use an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. We found that hypoxia altered the expression of over 2000 genes and that approximately 40% of these gene expression changes required FOXO. We discovered that hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, we identified homeodomain-interacting protein kinase (Hipk) as being required for hypoxia survival. We also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and we showed that hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, we discovered that FOXO was required for suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.
Two types of motor inhibition after action errors in humans
Authors: Guan Yao , Wessel Jan R.
Published: June 2022

Adaptive behavior requires the ability to appropriately react to action errors. Post-error slowing of response times (PES) is one of the most reliable phenomena in cognitive neuroscience. It has been proposed that PES is partially achieved through inhibition of the motor system. However, there is no direct evidence for this link – or indeed, that the motor system is physiologically inhibited after errors altogether. Here, we used transcranial magnetic stimulation and electromyography to measure cortico-spinal excitability (CSE) across four experiments using a Simon task, in which human participants sometimes committed errors. Errors were followed by reduced CSE at two different time points, and in two different modes. Shortly after error commission (250ms) CSE was broadly suppressed – i.e., even task-unrelated motor effectors were inhibited. During the preparation of the subsequent response, CSE was specifically reduced at task-related effectors only. This latter effect was directly related to PES, with stronger CSE suppression accompanying greater PES. This suggests that PES is achieved through increased inhibitory control during post-error responses. To provide converging evidence, we then re-analyzed an openly-available EEG dataset that contained both Simon- and Stop-signal tasks using independent component analysis. We found that the same neural source component that indexed action-cancellation in the stop-signal task also showed clear PES-related activity during post-error responses in the Simon task. Together, these findings provide clear evidence that post-error adaptation is partially achieved through motor inhibition. Moreover, inhibition is engaged in two modes (first non-selective, then selective), aligning with recent multi-stage theories of error processing.
Preclinical safety evaluation of continuous UV-A lighting in an operative setting
Authors: Guenter Rachael , Zheng-Pywell Rui , Herring Brendon , Murphy Madisen , Benner Kevin , Rose J. Bart
Published: June 2022

Background

Germicidal ultraviolet (UV-C) light has been shown as an effective modality for disinfection in laboratory settings and in the operative room. Traditionally, short-wavelength UV-C devices, which have previously been shown to cause DNA damage, are utilized only for disinfection in pre- and post-operative settings and are not continuously active during operations. Continuous use of intraoperative UV light can potentially decrease pathogens and subsequent surgical site infections (SSIs), which arise in approximately 5-15% of operative cases. SSIs are a significant determinant of patient morbidity, readmission rates, and overall cost. Therefore, a method of UV light disinfection with a low risk of DNA damage is needed so that greater antimicrobial protection can be afforded to patients during the entirety of their surgical procedures. A new disinfection device that harnesses longer-wavelength UV-A light to disinfect the surgical field throughout the entirety of the procedure, including pre- and post-operation, has been developed.

Methods

This study aimed to determine if intraoperatively administered UV-A light was safe, as defined by the minimal presence of DNA damage and safe amounts of reflection upon medical personnel. Using in vitro models, we examined the differential impacts of UV-C and UV-A light on DNA damage and repair pathways. In a murine model, we looked at the difference in production of DNA damage photoproducts between UV-A and UV-C exposure.

Results

Our results show UV-A light does not induce a significant amount of DNA damage at the cellular or tissue level. Furthermore, a preclinical porcine study showed that surgical personnel were exposed to safe levels of UV-A irradiance from an overhead UV-A light used during an operation. The amount of UV-A transmitted through surgical personal protective equipment (PPE) also remained within safe levels.

Conclusions

In conclusion, we found that UV-A may be a safe for intraoperative use.
Locus-specific enrichment analysis of 5-hydroxymethylcytosine reveals novel genes associated with breast carcinogenesis
Authors: Ramasamy Deepa , Rao Arunagiri Kuha Deva Magendhra , Balaiah Meenakumari , Rangan Arvinden Vittal , Sundersingh Shirley , Veluswami Sridevi , Thangarajan Rajkumar , Mani Samson
Published: June 2022

Background

An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development.

Results

We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer, which correlate strongly with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions such as the intergenic regions (>-5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples, positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, CNIH3, and BNIPL implicate a pro-oncogenic role in breast cancer. Therefore, 5-hmC modified regions could be used as promising diagnostic and therapeutic markers for breast cancer.

Conclusion

Global loss of 5-hmC is associated with down-regulation of the TET 1 and TET3 genes. Genome-wide profiling has revealed a profound imbalance in the region-specific distribution of 5-mC and 5-hmC in breast cancer. Predominant 5-hmC modifications are localized at distal gene regulatory sites. Novel 5-hmC candidates associated with breast cancer have been identified. Hence, these results provide new insights in the loci-specific accumulation of 5-mC and 5-hmC which are aberrantly methylated and demethylated in breast cancer.