bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune cell multi-omics analysis reveals contribution of oxidative phosphorylation to B cell functions and organ damage of lupus
Authors: Takeshima Yusuke , Iwasaki Yukiko , Nakano Masahiro , Narushima Yuta , Ota Mineto , Nagafuchi Yasuo , Sumitomo Shuji , Okamura Tomohisa , Elkon Keith B , Ishigaki Kazuyoshi , Suzuki Akari , Kochi Yuta , Yamamoto Kazuhiko , Fujio Keishi
Published: October 2021

Objective

Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease, with a poor long-term prognosis. The type I interferon (IFN) signature, a prominent feature of SLE, is not an ideal therapeutic target or outcome predictor. To explore immunological pathways in SLE more precisely, we performed integrative analysis of transcriptomics, epigenomics, and genomics using each immune cell subset from peripheral blood.

Methods

We sorted 18 immune cell subsets and identified the mRNA expression profiles and genetic polymorphisms in 107 SLE patients and 92 healthy controls. Open chromatin information was also taken by ATAC-seq analysis. Combined differentially expressed genes (DEGs) and expression quantitative trait loci (eQTL) analysis was conducted to find key driver genes in SLE pathogenesis.

Results

We found transcriptomic, epigenetic, and genetic importance of oxidative phosphorylation (OXPHOS)/mitochondrial dysfunction in SLE memory B cells. Particularly, we identified an OXPHOS-regulating gene, PRDX6, as a key driver in SLE B cells. Prdx6–deficient B cells showed upregulated mitochondrial respiration as well as antibody production. We revealed OXPHOS signature was associated with type I IFN signaling-related genes (ISRGs) signature in SLE memory B cells. Furthermore, the gene sets related to innate immune signaling among ISRGs presented correlation with OXPHOS and these two signatures showed associations with SLE organ damage as well as specific clinical phenotypes.

Conclusion

This work elucidated the potential prognostic marker for SLE. Since OXPHOS consists of the electron transport chain, a functional unit in mitochondria, these findings suggest the importance of mitochondrial dysfunction as a key immunological pathway involved in SLE.
A COMPARATIVE ANALAYSIS OF INTER-SITE GENE EXPRESSION HETEROGENICITY OF NORMAL HUMAN BUCCAL MUCOSA WITH NORMAL GINGIVAL MUCOSA
Authors: Rooban Thavarajah , Ranganathan Kannan
Published: October 2021

BACKGROUND

Description of heterogeneity of gene expression of various human intraoral sites are not adequate. The aim of this study was to explore the difference of gene expression profiles of whole tissue obtained from apparently normal human gingiva and buccal mucosa (HGM, HBM).

MATERIALS AND METHODS

Gene sets fulfilling inclusion and exclusion criteria of HGM and HBM in gene Expression Omnibus(GEO) database were identified, segregated, filtered and analysed using the ExAtlas online web tool using pre-determined cut-off. The differentially expressed genes were studied for epithelial keratinization related, housekeeping(HKG), extracellular matrix related(ECMRG) and epithelial-mesenchymal transition related genes(EMTRGs).

RESULTS

In all 40 HBM and 64 HGM formed the study group. In all there were 18012 significantly expressed genes. Of this, 1814 were over-expressed and 1862 under-expressed HBM genes as compared to HGM. One in five of all studied genes significantly differed between HBM and HGM. For the keratinization genes, 1 in 6 differed. One of every 5 HKG-proteomics genes differed between HBM and HGM, while this ratio was 1-in 4 for all ECMRGs and EMTRGs.

DISCUSSION

This difference in the gene expression between the HBM and HGM could possibly influence a multitude of biological pathways. This result could explain partly the difference in clinicopathological features of oral lesions occurring in HBM and HGM. The innate genotypic difference between the two intra-oral niches could serve as confounding factor in genotypic studies. Hence studies that compare the HBM and HGM should factor-in these findings while evaluating their results.
Lpnet: Reconstructing Phylogenetic Networks from Distances Using Integer Linear Programming
Authors: Guo Mengzhen , Grünewald Stefan
Published: October 2021

We present Lpnet, a variant of the widely used Neighbor-net method that approximates pairwise distances between taxa by a circular phylogenetic network. We first apply standard methods to construct a binary phylogenetic tree and then use integer linear programming to compute an optimal circular orderings that agrees with all tree splits. This approach achieves an improved approximation of the input distance for the clear majority of experiments that we have run for simulated and real data. We release an implementation in R that can handle up to 94 taxa and usually needs about one minute on a standard computer for 80 taxa. For larger taxa sets, we include a top-down heuristic which also tends to perform better than Neighbor-net.
HyINDEL – A Hybrid approach for Detection of Insertions and Deletions
Authors: Thatikunta Alok , Parekh Nita
Published: October 2021

Insertion and deletion (INDELs) mutations, the most common type of structural variation in the human genome, have been implicated in numerous human traits and diseases including rare genetic disorders and cancer. Next generation sequencing (NGS) technologies have drastically reduced the cost of sequencing whole genomes, greatly contributing to genome-wide detection of structural variants. However, due to large variations in INDEL sizes and presence of low complexity and repeat regions, their detection remains a challenge. Here we present a hybrid approach, HyINDEL, which integrates clustering, split-mapping and assembly-based approaches, for the detection of INDELs of all sizes (from small to large) and also identifies the insertion sequences. The method starts with identifying clusters of discordant and soft-clip reads which are validated by depth-of-coverage and alignment of soft-clip reads to identify candidate INDELs, while the assembly -based approach is used in identifying the insertion sequence. Performance of HyINDEL is evaluated on both simulated and real datasets and compared with state-of-the-art tools. A significant improvement in recall and F-score metrics as well as in breakpoint support is observed on using soft-clip alignments. It is freely available at https://github.com/alok123t/HyINDEL.
Trait-environment relationship in tadpoles of the southern Atlantic Forest
Authors: dos Santos Roseli Coelho , Brum Diego , Dalmolin Diego , Farina Renata Krentz , Lucas Elaine Maria , Tozetti Alexandro Marques
Published: October 2021

Environmental predictors select individuals by their functional traits, shaping the anuran assembly patterns. Individuals respond to environmental filters that can be on a local or regional scale.

In this study, we investigated the association between local (water and microhabitat) and landscape variables and the morphological traits of tadpoles of ponds and streams. The study was conducted in the southern region of the Brazilian Atlantic Forest. We sampled 28 waterbodies and recorded 22 anurans species. We performed RLQ and fourth-corner analyses to determine the patterns of trait-environment relationships and determine which environmental and landscape variables influence the morphological characteristics of tadpoles from streams and ponds.

We found that the morphological traits of tadpoles are influenced mainly by physicochemical and microhabitat attributes, being distinct between ponds and streams. In ponds, water depth, pH, and the presence of vegetation influence the morphological traits of the tadpoles, while in the streams water pH, temperature, conductivity, total alkalinity, Alk HCO3, and microhabitat variables played a major role in defining the traits.

Our results indicate that local components of habitat (water characteristics and microhabitat) influence functional traits of tadpoles in both ponds and streams, especially those supposedly related to locomotory, foraging and prey-detection abilities.
Anti-cancer potential of cannabis terpenes in a taxol-resistant model of breast cancer
Authors: Tomko Andrea M. , Whynot Erin G. , O’Leary Lauren F. , Dupré Denis J.
Published: October 2021

Chemotherapeutic resistance can limit breast cancer outcomes; therefore, the exploration of novel therapeutic options is warranted. Isolated compounds found in cannabis have previously been shown to exhibit anti-cancer effects, but little is known about their effects in resistant breast cancer. Our study aims to evaluate the effects of terpenes found in cannabis in in vitro chemotherapy-resistant model of breast cancer. We aimed to identify whether five terpenes found in cannabis produced anti-cancer effects, and if their effects were improved upon co-treatment with cannabinoids and flavonoids also found in cannabis. Nerolidol and β-caryophyllene produced the greatest cytotoxic effects, activated the apoptotic cascade and reduced cellular invasion. Combinations with the flavonoid kaempferol potentiated the cytotoxic effects of ocimene, terpinolene, and β-myrcene. Combinations of nerolidol and Δ9-tetrahydrocannabinol or cannabidiol produced variable responses ranging from antagonism and additivity to synergy, depending on concentrations used. Our results indicate that cannabis terpenes, alone or combined with cannabinoids and flavonoids, produced anti-cancer effects in chemotherapy-resistant breast cancer cell lines. This study is a first step in the identification of compounds that could have therapeutic potential in the treatment of resistant breast cancer.
Precise control of microtubule disassembly in living cells
Authors: Liu Grace Y. , Chen Shiau-Chi , Shaiv Kritika , Hong Shi-Rong , Yang Wen-Ting , Huang Shih-Han , Chang Ya-Chu , Cheng Hsuan , Lin Yu-Chun
Published: October 2021

Microtubules (MTs) are components of the evolutionarily conserved cytoskeleton, which tightly regulates various cellular activities. Our understanding of MTs is largely based on MT-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific MT populations due to their slow effects on the entire pool of MTs in cells. To address this limitation, we have used chemogenetics and optogenetics to disassemble specific MT subtypes by rapid recruitment of engineered MT-cleaving enzymes. Acute MT disassembly swiftly halted vesicular trafficking and lysosome dynamics. We also used this approach to disassemble MTs specifically modified by tyrosination and several MT-based structures including primary cilia, mitotic spindles, and intercellular bridges. These effects were rapidly reversed by inhibiting the activity or MT association of the cleaving enzymes. The disassembly of targeted MTs with spatial and temporal accuracy enables to uncover new insights of how MTs precisely regulate cellular architectures and functions.
Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility
Authors: Magallon Jesus , Vu Peter , Reeves Craig , Kwan Stella , Phan Kimberly , Oakley-Havens Crista L. , Rocha Kenneth , Jimenez Veronica , Ramirez María Soledad , Tolmasky Marcelo E.
Published: October 2021

Resistance to amikacin in Gram-negatives is usually mediated by the 6′-N-acetyltransferase type Ib [AAC(6′)-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6′ position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res. 2008, 68:5318-5325) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6′)-Ib, an enzyme commonly found in the clinics.
Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma
Authors: Femel Julia , Booth Jamie L. , Asnaashari Tina G. , Leachman Sancy A. , Tsujikawa Takahiro , White Kevin P. , Chang Young H. , Lund Amanda W.
Published: October 2021

Purpose

Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma.

Experimental Design

We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas.

Results

Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates.

Conclusions

We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for tumor cell exit (metastasis) or leukocyte trafficking (immune response).

This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

TRANSLATIONAL RELEVANCE

This report describes a quantitative, image-based method to investigate the relationship between the tumor-associated lymphovasculature and immune landscape in treatment naïve, primary human melanoma. The research shows that melanoma-associated blood and lymphatic vessels display context-dependent phenotypes that associate both with the risk of regional progression and immune infiltration. These findings indicate that stromal/vascular heterogeneity may underlie regional differences in immunogenicity and thus present opportunities for future biomarker development and therapeutic intervention.
Broad-scale puma connectivity could restore genomic diversity to fine-scale coastal populations
Authors: Gustafson Kyle D. , Gagne Roderick B. , Buchalski Michael R. , Vickers T. Winston , Riley Seth P.D. , Sikich Jeff , Rudd Jaime L. , Dellinger Justin A. , LaCava Melanie E.F. , Ernest Holly B.
Published: October 2021

Urbanization is decreasing wildlife habitat and connectivity worldwide, including for apex predators, such as the puma (Puma concolor). Puma populations along California’s central and southern coastal habitats have experienced rapid fragmentation from development, leading to calls for demographic and genetic management. To address urgent conservation genomic concerns, we used double-digest restriction-site associated DNA (ddRAD) sequencing to analyze 16,285 genome-wide single-nucleotide polymorphisms (SNPs) from 401 broadly sampled pumas. Our analyses indicated support for 4–10 geographically nested, broad- to fine-scale genetic clusters. At the broadest scale, the 4 genetic clusters had high genetic diversity and exhibited low linkage disequilibrium, indicating pumas have retained statewide genomic diversity. However, multiple lines of evidence indicated substructure, including 10 fine-scale genetic clusters, some of which exhibited allelic fixation and linkage disequilibrium. Fragmented populations along the Southern Coast and Central Coast had particularly low genetic diversity and strong linkage disequilibrium, indicating genetic drift and close inbreeding. Our results demonstrate that genetically at-risk populations are typically nested within a broader-scale group of interconnected populations that collectively retains high genetic diversity and heterogeneous fixations. Thus, extant variation at the broader scale has potential to restore diversity to local populations if management actions can enhance vital gene flow and recombine locally sequestered genetic diversity. These state- and genome-wide results are critically important for science-based conservation and management practices. Our broad- and fine-scale population genomic analysis highlights the information that can be gained from population genomic studies aiming to provide guidance for fragmented population conservation management.
Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function
Authors: Raut Prakash , Weller Sasha R. , Obeng Bright , Soos Brandy L. , West Bailey E. , Potts Christian M. , Sangroula Suraj , Kinney Marissa S. , Burnell John E. , King Benjamin L. , Gosse Julie A. , Hess Samuel T.
Published: October 2021

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 μM, 1 hour) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.
Convergent evolution of polyploid genomes from across the eukaryotic tree of life
Authors: Hao Yue , Fleming Jonathon , Petterson Joanna , Lyons Eric , Edger Patrick P. , Pires Chris , Thorne Jeffrey L. , Conant Gavin C.
Published: October 2021

By modeling the homoeologous gene losses that occurred in fifty genomes deriving from ten distinct polyploidy events, we show that the evolutionary forces acting on polyploids are remarkably similar, regardless of whether they occur in flowering plants, ciliates, fishes or yeasts. The models suggest these events were nearly all allopolyploidies, with two distinct progenitors contributing to the modern species. We show that many of the events show a relative rate of duplicate gene loss prior to the first post-polyploidy speciation that is significantly higher than in later phases of their evolution. The relatively low selective constraint seen for the single-copy genes these losses produced lead us to suggest that most of the purely selectively neutral duplicate gene losses occur in the immediate post-polyploid period. We also find ongoing and extensive reciprocal gene losses (RGL; alternative losses of duplicated ancestral genes) between these genomes. With the exception of a handful of closely related taxa, all of these polyploid organisms are separated from each other by tens to thousands of reciprocal gene losses. As a result, it is very unlikely that viable diploid hybrid species could form between these taxa, since matings between such hybrids would tend to produce offspring lacking essential genes. It is therefore possible that the relatively high frequency of recurrent polyploidies in some lineages may be due to the ability of new polyploidies to bypass RGL barriers.
Self-grooming promotes social attraction in mice via chemosensory communication
Authors: Zhang Yun-Feng , Janke Emma , Bhattarai Janardhan P. , Wesson Daniel W. , Ma Minghong
Published: October 2021

Self-grooming is a stereotyped behavior displayed by nearly all animals. Among other established functions, self-grooming is implicated in social communication in some animals. However, whether self-grooming specifically influences behaviors of nearby individuals has not been directly tested, partly due to the technical challenge of inducing self-grooming in a reliable and temporally controllable manner. We recently found that optogenetic activation of dopamine D3 receptor expressing neurons in the ventral striatal islands of Calleja robustly induces orofacial grooming in mice. Using this optogenetic manipulation, here we demonstrate that observer mice exhibit social preference for mice that groom more regardless of biological sex. Moreover, grooming-induced social attraction depends on volatile chemosensory cues broadcasted from grooming mice. Collectively, our study establishes self-grooming as a means of promoting social attraction among mice via volatile cues, suggesting an additional benefit for animals to allocate a significant amount of time to this behavior.
Islet transplantation tolerance in animals with defined histocompatibility and diabetes
Authors: Bhagchandani Preksha , Chang Charles A. , Zhao Weichen , Ghila Luiza , Herrera Pedro L. , Chera Simona , Kim Seung K.
Published: October 2021

Advances in organ transplantation benefit from development of genetically inbred animal strains with defined histocompatibility and cell-specific markers to distinguish donor and host cell subsets. For studies of pancreatic islet transplantation tolerance in diabetes, an invariant method to ablate host β cells and induce diabetes would provide an immense additional advantage. Here we detail development and use ofB6 RIP-DTRmice, an immunocompetent line permitting diabetes induction with 100% penetrance. This inbred line is homozygous for the C57BL/6J major histocompatibility complex (MHC) haplotype and expresses the mutantCD45.1allele in the hematopoietic lineage. β cell-specific expression of a high-affinity receptor for diphtheria toxin (DT) permits experimental β cell ablation and diabetes induction after DT administration. Diabetes reversal for over one year was achieved after transplantation with congenic C57BL/6J islets, but not with MHC-mismatched BALB/c islets, which were rapidly rejected. In summary, the generation of a C57BL/6J congenic line harboring theCD45.1allele andIns2-HBEGFtransgene should advance studies of islet transplantation tolerance and mechanisms to improve islet engraftment and function, thereby optimizing development of cell replacement strategies for diabetes mellitus.
Wind and route choice affect performance of bees flying above versus within a cluttered obstacle field
Authors: Burnett Nicholas P. , Badger Marc A. , Combes Stacey A.
Published: October 2021

Bees flying through natural landscapes encounter physical challenges, such as wind and cluttered vegetation. The influence of these factors on the flight performance of bees remains unknown. We analyzed 548 videos of wild-caught honeybees (Apis mellifera) flying through an enclosure containing a field of vertical obstacles that bees could fly within (through open corridors, without maneuvering) or above. We examined how obstacle field height, wind presence and direction (headwinds or tailwinds) affected altitude, ground speed, and side-to-side casting (lateral excursions) of bees. When obstacle fields were short, bees flew at altitudes near the midpoint between the tunnel floor and ceiling. When obstacle fields approached or exceeded this midpoint, bees typically, but not always, increased their altitudes to fly over the obstacles. Bees that flew above the obstacle fields exhibited 40% faster ground speeds and 36% larger lateral excursions than bees that flew within the obstacle fields, likely due to the visual feedback from obstacles and narrow space available within the obstacle field. Wind had a strong effect on ground speed and lateral excursions, but not altitude. Bees flew 12-19% faster in tailwinds than in the other wind conditions, but their lateral excursions were 19% larger in any wind, regardless of its direction, than in still air. Our results show that bees flying through complex environments display flexible flight behaviors (e.g., flying above versus within obstacles), which affect flight performance. Similar choices in natural landscapes could have broad implications for foraging efficiency, pollination, and mortality in wild bees.
Inferring the mode and strength of ongoing selection
Authors: Barroso Gustavo V. , Lohmueller Kirk E.
Published: October 2021

Genome sequence data is no longer scarce. The UK Biobank alone comprises 200,000 individual genomes, with more on the way, leading the field of human genetics towards sequencing entire populations. Within the next decades, other model organisms will follow suit, especially domesticated species such as crops and livestock. Having sequences from most individuals in a population will present new challenges for using these data to improve health and agriculture in the pursuit of a sustainable future. Existing population genetic methods are designed to model hundreds of randomly sampled sequences, but are not optimized for extracting the information contained in the larger and richer datasets that are beginning to emerge, with thousands of closely related individuals. Here we develop a new method called TIDES (Trio-based Inference of Dominance and Selection) that uses data from tens of thousands of family trios to make inferences about natural selection acting in a single generation. TIDES further improves on the state-of-the-art by making no assumptions regarding demography, linkage or dominance. We discuss how our method paves the way for studying natural selection from new angles.
An intein-mediated split–nCas9 system for base editing in plants
Authors: Yuan Guoliang , Lu Haiwei , Hassan Md Mahmudul , Liu Yang , Li Yi , Abraham Paul E. , Tuskan Gerald A. , Yang Xiaohan
Published: October 2021

Virus-assisted delivery of the clustered regularly interspaced short palindromic (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. However, the relatively large size of the CRISPR/Cas9 system is challenging to package into viral vectors with confined packaging capacity. To address this technical challenge, we developed a strategy that splits the required CRISPR-Cas9 components across a dual-vector system in which CRISPR-Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by successfully demonstrating split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-SpnCas9 is capable of inducing efficient base editing in plant cells and identified several valid split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.
Cheating the cheater: Suppressing false positive enrichment during biosensor-guided biocatalyst engineering
Authors: Trivedi Vikas D. , Mohan Karishma , Chappell Todd C. , Mays Zachary J. S. , Nair Nikhil U.
Published: October 2021

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection, LOD) suppresses cheater population enrichment. Further we show that, if we couple the biosensor-based screen with an orthogonal pre-screen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting (FACS). Using the approach developed here, we were successfully able to isolate PAL variants with ~70% high kcat after a single sort. These mutants have tremendous potential in Phenylketonuria (PKU) treatment and flavonoid production.
In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris
Authors: Rybak Jeffrey M. , Barker Katherine S. , Muñoz José F. , Parker Josie E. , Ahmad Suhail , Mokaddas Eiman , Abdullah Aneesa , Elhagracy Rehab S. , Kelly Steve L. , Cuomo Christina A. , Rogers P. David
Published: October 2021

Candida auris has emerged as a healthcare-associated and multidrug-resistant fungal pathogen of great clinical concern. While as much as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, to date, no mechanisms contributing to this resistance have been identified. We report here mutations in the C. auris sterol-methyltransferase gene, ERG6, as the first identified mechanism of amphotericin B resistance in this emerging pathogen and describe the clinical case in which this high-level amphotericin B resistance was acquired in vivo during therapy. Whole genome sequencing revealed the four C. auris isolates obtained from this single patient case to be genetically related and identified a mutation in ERG6 as being associated with amphotericin B resistance. Cas9-mediated genetic manipulations confirmed this mutation alone to confer a >32-fold increase in amphotericin B resistance, and comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harboring the clinically derived ERG6 mutation. Together these findings represent the first significant step forward in the understanding of clinical amphotericin B resistance in C. auris.
Mutagenesis-Visualization: Analysis of Site-Saturation Mutagenesis Datasets in Python
Authors: Hidalgo Frank , Templeton Sage , Gallegos Che Olavarria , Wang Joanne
Published: October 2021

Site-saturation mutagenesis experiments have been transformative in our study of protein function. Despite the rich data generated from such experiments, current tools for processing, analyzing, and visualizing the data offer only a limited set of static visualization tools that are difficult to customize. Furthermore, usage of the tools requires extensive experience and programming knowledge, slowing the research process for those in the biological field who are unfamiliar with programming. Here, we introducemutagenesis-visualization, a Python package for creating publication-quality figures for site-saturation mutagenesis datasets without the need for prior Python or statistics experience, where each of the graphs is generated with a one-line command. The plots can be rendered as nativeMatplotlibobjects (easy to stylize) orPlotlyobjects (interactive graphs). Additionally, the software offers the possibility to visualize the datasets onPymol.

Availability and implementation

The software can be installed fromPyPIorGitHubusing thepippackage manager and is compatible with Python ≥ 3.8. Thedocumentationcan be found atreadthedocsand thesource codeonGitHub.
Identification of Iron-Sulfur (Fe-S) and Zn-binding Sites Within Proteomes Predicted by DeepMind’s AlphaFold2 Program Dramatically Expands the Metalloproteome
Authors: Wehrspan Zachary J. , McDonnell Robert T. , Elcock Adrian H.
Published: October 2021

DeepMind’s AlphaFold2 software has ushered in a revolution in high quality, 3D protein structure prediction. In very recent work by the DeepMind team, structure predictions have been made for entire proteomes of twenty-one organisms, with >360,000 structures made available for download. Here we show that thousands of novel binding sites for iron-sulfur (Fe-S) clusters and zinc ions can be identified within these predicted structures by exhaustive enumeration of all potential ligand-binding orientations. We demonstrate that AlphaFold2 routinely makes highly specific predictions of ligand binding sites: for example, binding sites that are comprised exclusively of four cysteine sidechains fall into three clusters, representing binding sites for 4Fe-4S clusters, 2Fe-2S clusters, or individual Zn ions. We show further: (a) that the majority of known Fe-S cluster and Zn-binding sites documented in UniProt are recovered by the AlphaFold2 structures, (b) that there are occasional disputes between AlphaFold2 and UniProt with AlphaFold2 predicting highly plausible alternative binding sites, (c) that the Fe-S cluster binding sites that we identify in E. coli agree well with previous bioinformatics predictions, (d) that cysteines predicted here to be part of Fe-S cluster or Zn-binding sites show little overlap with those shown via chemoproteomics techniques to be highly reactive, and (e) that AlphaFold2 occasionally appears to build erroneous disulfide bonds between cysteines that should instead coordinate a ligand. These results suggest that AlphaFold2 could be an important tool for the functional annotation of proteomes, and the methodology presented here is likely to be useful for predicting other ligand-binding sites.
Mistic: an open-source multiplexed image t-SNE viewer
Authors: Prabhakaran Sandhya , Gatenbee Chandler , Robertson-Tessi Mark , West Jeffrey , Beg Amer A. , Gray Jhanelle , Antonia Scott , Gatenby Robert A. , Anderson Alexander R. A.
Published: October 2021

Understanding the complex ecology of a tumor tissue and the spatio-temporal relationships between its cellular and microenvironment components is becoming a key component of translational research, especially in immune-oncology. The generation and analysis of multiplexed images from patient samples is of paramount importance to facilitate this understanding. In this work, we present Mistic, an open-source multiplexed image t-SNE viewer that enables the simultaneous viewing of multiple 2D images rendered using multiple layout options to provide an overall visual preview of the entire dataset. In particular, the positions of the images can be taken from t-SNE or UMAP coordinates. This grouped view of all the images further aids an exploratory understanding of the specific expression pattern of a given biomarker or collection of biomarkers across all images, helps to identify images expressing a particular phenotype or to select images for subsequent downstream analysis. Currently there is no freely available tool to generate such image t-SNEs. Mistic is open-source and can be downloaded at:https://github.com/MathOnco/Mistic.
The duration of caffeine treatment plays an essential role in its effect on sleep and circadian rhythm
Authors: Segu Aishwarya , Kannan Nisha N
Published: October 2021

Sleep is regulated by the homeostatic system and the circadian clock. The circadian clock governs the timing of sleep-wake cycles and the homeostatic mechanisms modulate the amount and depth of sleep. Caffeine intake promotes wakefulness inDrosophila. In humans, caffeine is consumed on a daily basis and hence it is important to understand the effect of prolonged caffeine intake on both circadian and homeostatic regulation of sleep. Furthermore, sleep changes with age and the impact of caffeine on age dependent sleep fragmentation is yet to be understood. Hence in the present study, we examined the effect of short exposure to caffeine on homeostatic sleep and age dependent sleep fragmentation inDrosophila. We further assessed the effect of prolonged exposure to caffeine on homeostatic sleep and circadian clock inDrosophila. The results of our study showed that short exposure to caffeine reduces sleep in flies and enhances sleep fragmentation with increasing age. On the other hand, prolonged caffeine exposure did not exert any significant effect on duration of sleep in mature flies. Nevertheless, prolonged caffeine ingestion decreased the morning and evening anticipatory activity in these flies indicating that it affects the circadian rhythm. These flies also exhibited phase delay in the clock genetimelesstranscript oscillation and altered behavioral rhythm with either a longer free running period or arrhythmicity under constant darkness. In summary, the results of our studies showed that short exposure to caffeine increases the sleep fragmentation with age whereas prolonged caffeine exposure disrupts the circadian clock.

Statement of Significance

In the present study, we assessed the effect of duration of caffeine intake on age dependent sleep fragmentation and circadian clock by usingDrosophilaas a model organism. We found that short exposure to caffeine reduced sleep and food intake in flies. It also increased the sleep fragmentation with age. On the other hand prolonged caffeine exposure did not affect the sleep and feeding indicating that these flies developed tolerance to caffeine. As in mammals, caffeine slows down the circadian clock in flies. Most importantly, prolonged caffeine treatment disrupted the circadian rhythm inDrosophila. Our studies provide new insights into the differential effect of duration of caffeine intake on circadian clock and sleep.
Loop 1 of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation but not the rate of ADP release
Authors: Gargey Akhil , Nesmelov Yuri E.
Published: October 2021

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450 was previously discovered in the molecular dynamics simulations. It was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulates ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutant of the myosin head construct (1–843 amino acid residues) were expressed in differentiated C2C12 cells, and the kinetics of ATP induced actomyosin dissociation and ADP release were characterized using transient kinetics spectrophotometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. We observed no effect of loop 1 flexibility on the rate of ADP release from actomyosin.

Highlights

Human cardiac myosin has the isoform-specific salt bridge, electrostatically linking loop 1 and myosin head backbone.

Absence of loop 1 – backbone salt bridge increases rate of ATP-induced actomyosin dissociation

The rate of ADP dissociation from actomyosin is not affected by the isoform-specific salt bridge.
Functional Effects of Four or Fewer Critical Genes Linked to Lung Cancers and New Subtypes Detected by A New Machine Learning Classifier
Authors: Zhang Zhengjun
Published: October 2021

Finding genes biologically directly or indirectly related to lung cancer has been drawing much attention, and many genes directly related to lung cancer have been reported. However, it has not been confirmed whether those published 'key' genes are truly critical to lung cancer formation, i.e., they may be with very limited useful information. As a result, finding essential genes remains a challenging lung cancer research problem. Using a recently developed competing linear factor analysis method in differentially expressed gene detection, we advance the study of lung cancer critical genes detection to a uniformly informative level. A set of common four genes and their functional effects are detected to be differentially expressed in tumor and non-tumor samples with 100% sensitivity and 100% specificity in one study of lung adenocarcinoma (LUAD) and one study of squamous cell lung cancers (LUSC) (two North American cohorts with 20429 genes, 576 and 552 samples respectively). Two additional analyses also gain accuracy of 97.8% sensitivity and 100% specificity in one study of non-small cell lung carcinomas (NSCLC, a European cohort with 20356 genes and 156 samples), and an accuracy of 100% sensitivity and 95% specificity (1 out of 20 non-tumor samples) in one study of ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas (LUAD, a Japanese cohort with 20356 genes and 224 samples). There are some common genes, but different functional effects, within each set of four genes among two North American cohorts and a European cohort and among North American cohorts and the Japanese cohort. These results show the four-gene-based classifiers are robust with different types of lung cancers and different race cohorts and accurate. The functional effects of four genes disclose significantly other mechanisms (mysteries) between LUAD and LUSC. These sets of four genes and their functional effects are considered to be essential for lung cancer studies and practice. These genes' functional effects naturally classify patients into different groups (more than seven subtypes). Subtype information is useful for personalized therapies. The new findings can motivate new lung cancer research in more focused and targeted directions to save lives, protect people, and reduce enormous economic costs in research and lung cancer treatments.
Reliability and Stability Challenges in ABCD Task fMRI Data
Authors: Kennedy James T. , Harms Michael P. , Korucuoglu Ozlem , Astafiev Serguei V. , Barch Deanna M. , Thompson Wesley K. , Bjork James M. , Anokhin Andrey P.
Published: October 2021

Trait stability of measures is an essential requirement for individual differences research. Functional MRI has been increasingly used in studies that rely on the assumption of trait stability, such as attempts to relate task related brain activation to individual differences in behavior and psychopathology. However, recent research using adult samples has questioned the trait stability of task-fMRI measures, as assessed by test-retest correlations. To date, little is known about trait stability of task fMRI in children. Here, we examined within-session reliability and longitudinal stability of task-fMRI using data from the Adolescent Brain Cognitive Development (ABCD) Study using its tasks focused on reward processing, response inhibition, and working memory. We also evaluated the effects of factors potentially affecting reliability and stability. Reliability and stability [quantified via an intraclass correlation (ICC) that focuses on rank consistency] was poor in virtually all brain regions, with an average ICC of .078 and .054 for short (within-session) and long-term (between-session) ICCs, respectively, in regions of interest (ROIs) historically-recruited by the tasks. ICC values in ROIs did not exceed the ‘poor’ cut-off of .4, and in fact rarely exceeded .2 (only 5.9%). Motion had a pronounced effect on estimated ICCs, with the lowest motion quartile of participants having a mean reliability/stability three times higher (albeit still ‘poor’) than the highest motion quartile. Regions with stronger activation tended to show higher ICCs, with the absolute value of activity and reliability/stability correlating at .53. Across regions, the magnitude of age-related longitudinal (between-session) changes positively correlated with the longitudinal stability of individual differences, which suggests developmental change was not necessarily responsible for poor stability. Poor reliability and stability of task-fMRI, particularly in children, diminishes potential utility of fMRI data due to a drastic reduction of effect sizes and, consequently, statistical power for the detection of brain-behavior associations. This essential issue needs to be addressed through optimization of preprocessing pipelines and data denoising methods.