Biology subjects

Xu, D.

Publications and source records attributed to Xu, D..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

Protein nonadditive expression and solubility in Arabidopsis hybrids and allotetraploids

Interspecific hybridization in plants often leads to allopolyploids including most important crops such as wheat, cotton, and canola, while many other crops such as corn and sorghum are grown as hybrid. Both allopolyploids and hybrids show hybrid vigor or heterosis. The phenomenon has been widely applied in agriculture and extensively studied at the genetic and gene expression levels. However, proteomic changes in hybrids and polyploids remain poorly understood. Here, we report comparative analysis of soluble and insoluble proteomes in Arabidopsis intraspecific and interspecific hybrids or allotetraploids formed between A. thaliana and A. arenosa. Both allotetraploids and intraspecific hybrids displayed nonadditive expression (unequal to the sum of the two parents) of the proteins, most of which were involved in biotic and abiotic stress responses. In the allotetraploids, homoeolog-expression bias was not observed among all proteins examined but could occur among 17-20% of the nonadditively expressed proteins, consistent with the transcriptome results. Among expression-biased homoeologs, there were more A. thaliana-biased than A. arenosa-biased homoeologs. Analysis of the insoluble and soluble proteomes revealed more soluble proteins in the hybrids than their parents but not in the allotetraploids. Most proteins in ribosomal biosynthesis and in the thylakoid lumen, membrane, and stroma were in the soluble fractions, indicating a role of protein stability in photosynthetic activities for promoting growth. These results collectively support roles of nonadditive expression of stress-responsive proteins and increased solubility of photosynthetic proteins in Arabidopsis hybrids and allotetraploids, which may promote hybrid vigor. Plain Language SummaryWe analyzed fractionated proteomes to test protein abundance and solubility in Arabidopsis hybrids and polyploids. Many proteins in stress-responses are nonadditively expressed in intraspecific hybrids or allotetraploids. There are more soluble proteins of ribosomal biosynthesis and photosynthetic activities in the hybrids than in their parents but not in allotetraploids. Expression levels are equal among most protein homoeologs in the allotetraploids but are biased for ~20% of nonadditively expressed proteins. Nonadditive protein expression and solubility may play a role in heterosis.

genetics↗

bioRxiv2023

A non-canonical function of LDHB promotes SLC7A11-mediated glutathione metabolism and protects against glutaminolysis-dependent ferroptosis in KRAS-driven lung cancer

Ferroptosis, a form of non-apoptotic cell death program driven by excessive lipid peroxidation and an important mechanism of tumor suppression, is frequently dysregulated in cancer. However, the mechanisms underlying impaired ferroptosis in oncogene-specific tumors remain poorly understood. Here we report a non- canonical role of lactate dehydrogenase B (LDHB), whose main activity is the conversion of lactate to pyruvate, in protecting KRAS-mutated lung cancer from ferroptosis. Silencing of LDHB impairs intracellular glutathione (GSH) metabolism and drives the hypersensitivity of KRAS-mutant cells to ferroptosis inducers by inhibiting the SLC7A11/GSH/GPX4 axis, a central antioxidant system against lipid peroxidation and ferroptosis by catalyzing GSH synthesis and utilization. Mechanistically, LDHB promotes SLC7A11 expression and GSH biosynthesis, and inhibition of LDHB confers metabolic synthetic lethality with ferroptosis inducers due to increased glutaminolysis and production of reactive oxygen species (ROS) in mitochondria, ultimately triggering ferroptosis of KRAS-driven lung cancer cells. Consequently, combined inhibition of LDHB and SLC7A11 synergistically suppresses tumor growth in multiple KRAS-mutant lung cancer implants and in an autochthonous model of Kras-induced lung adenocarcinoma. Taken together, our results reveal a hitherto unrecognized mechanism of ferroptosis defense by glycolytic LDHB and suggest a new strategy for the treatment of KRAS-dependent lung cancer.

cancer biology↗

bioRxiv2023

rRNA intermediates coordinate the multilayered nucleolar phase transition in C. elegans

The nucleolus is the most prominent membraneless organelle within the nucleus and plays essential roles in rRNA transcription and processing and ribosome assembly. How the structure of the nucleolus is maintained and regulated is poorly understood. Here, we identified two types of nucleoli in C. elegans. Type I nucleoli are spherical, and rRNA transcription and processing factors are evenly distributed throughout the nucleolus. In type II nucleoli, rRNA transcription and processing factors exclusively accumulate in the periphery rim, which is named the nucleolar ring. The hollow vacuole inside the nucleolar ring contains proteins that usually localize in the nucleoplasm but are capable of exchanging contents across the ring. The high-order structure of the nucleolus is dynamically regulated in C. elegans. Faithful rRNA processing is important to maintain the spherical structure of the nucleoli. The depletion of a class of rRNA processing factors, for example, class I ribosomal proteins of the large subunit (RPL), which are involved in 27SA2 rRNA processing, reshaped spherical nucleoli to a ring-shaped nucleolar structure. The inhibition of RNAP I transcription and depletion of two conserved nucleolar factors, nucleolin and fibrillarin, prohibits the formation of the nucleolar ring. We concluded that the integrity of nucleoli is highly dependent on rRNA processing and maturation, which may provide a mechanism to coordinate structure maintenance and gene expression.

cell biology↗

bioRxiv2022

Single-cell Sequencing Highlights Heterogeneity and Malignant Progression in Actinic Keratosis and Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent of the keratinocyte-derived malignancies with actinic keratosis (AK) as a precancerous lesion. To comprehensively delineate the underlying mechanisms for the whole progression from normal skin to AK to invasive cSCC, we performed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomes of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC and their matched normal tissues, covering comprehensive clinical courses of cSCC. We identified diverse cell types, including important subtypes with different gene expression profiles and functions in major keratinocytes. In SCCIS, we discovered the malignant subtypes of basal cells with differential proliferative and migration potential. Differentially expressed genes (DEGs) analysis screened out multiple key driver genes including transcription factors (TFs) along AK to cSCC progression. Immunohistochemistry (IHC) / immunofluorescence (IF) experiments and single-cell ATAC sequencing (scATAC-seq) data verified the expression changes of these genes. The functional experiments confirmed the important roles of these genes in regulating cell proliferation, apoptosis, migration and invasion in cSCC tumor. Furthermore, we comprehensively described the tumor microenvironment (TME) landscape and potential keratinocyte-TME crosstalk in cSCC providing theoretical basis for immunotherapy. Together, our findings provide a valuable resource for deciphering the progression from AK to cSCC and identifying potential targets for anticancer treatment of cSCC.

cancer biology↗

bioRxiv2022

Designing epitope-focused vaccines via antigen reorientation

A major challenge in vaccine development, especially against rapidly evolving viruses, is the ability to focus the immune response toward evolutionarily conserved antigenic regions to confer broad protection. For example, while many broadly neutralizing antibodies against influenza have been found to target the highly conserved stem region of hemagglutinin (HA-stem), the immune response to seasonal influenza vaccines is predominantly directed to the immunodominant but variable head region (HA-head), leading to narrow-spectrum efficacy. Here, we first introduce an approach to controlling antigen orientation based on the site-specific insertion of short stretches of aspartate residues (oligoD) that facilitates antigen-binding to alum adjuvants. We demonstrate the generalizability of this approach to antigens from the Ebola virus, SARS-CoV-2, and influenza and observe enhanced antibody responses following immunization in all cases. Next, we use this approach to reorient HA in an "upside down" configuration, which we envision increases HA-stem exposure, therefore also improving its immunogenicity compared to HA-head. When applied to HA of H2N2 A/Japan/305/1957, the reoriented H2 HA (reoH2HA) on alum induced a stem-directed antibody response that cross-reacted with both group 1 and 2 influenza A HAs. Our results demonstrate the possibility and benefits of antigen reorientation via oligoD insertion, which represents a generalizable immunofocusing approach readily applicable for designing epitope-focused vaccine candidates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/521291v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@185f949org.highwire.dtl.DTLVardef@b132a9org.highwire.dtl.DTLVardef@174fcc8org.highwire.dtl.DTLVardef@17fb872_HPS_FORMAT_FIGEXP M_FIG C_FIG Seasonal influenza vaccines induce a biased antibody response against the variable head of hemagglutinin, whereas conserved epitopes on the stem are a target for universal vaccines. Here we show that reorienting HA in an "upside-down" configuration sterically occludes the head and redirects the antibody response to the more exposed stem, thereby inducing broad cross-reactivity against hemagglutinins from diverse influenza strains.

immunology↗

bioRxiv2022

Inhibition of proline tyrosine kinase 2 (Pyk2) phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohns Disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease through genome wide association studies. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while an inhibitor of Pyk2, PF-431396 hydrate, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

microbiology↗

bioRxiv2022

A ZTF-7/RPS-2 complex mediates the cold-warm response in C. elegans

Temperature greatly affects numerous biological processes in all organisms. How multicellular organisms respond to and are impacted by hypothermic stress remains elusive. Here, we found that cold-warm stimuli induced depletion of the RNA exosome complex in the nucleoli but enriched it in the nucleoplasm. To further understand the function and mechanism of cold-warm stimuli, we conducted forward genetic screening and identified ZTF-7, which is required for RNA exosome depletion from nucleoli upon transient cold-warm exposure in C. elegans. ZTF-7 is a putative ortholog of human ZNF277 that may contribute to language impairments. Immunoprecipitation followed by mass spectrometry (IP-MS) found that ZTF-7 interacted with RPS-2, which is a ribosomal protein of the small subunit and participates in pre-rRNA processing. A partial depletion of RPS-2 and other proteins of the small ribosomal subunit blocked the cold-warm stimuli-induced reduction of exosome subunits from the nucleoli. These results established a novel mechanism by which C. elegans responds to environmental cold-warm exposure.

genetics↗

bioRxiv2022

TPL2 kinase activity regulates microglial inflammatory responses and promotes neurodegeneration in tauopathy mice

TPL2 (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNAseq analysis indicates protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.

neuroscience↗

bioRxiv2022

CIDP: A multi-functional platform for designing CRISPR sgRNAs

Most of sgRNA-design tools can be run under the precondition of the choice of closely-related species. However, it is hard to select an ideal closely-related species, as more and more different species were studied, and this situation was particular seriously in plant studies. Here, we introduced a new software, CRISPR Integrated Design Platform (CIDP), to solve the problem by allowing users to input genomic sequences for designing sgRNAs. The main function of CIDP was to design sgRNAs after building the sgRNA database using the input genomic sequences. Furthermore, in order to minimize the off-target effects, CIDP will search sgRNAs that appear only once across the whole genome on the target sequences. Meanwhile, CIDP set relevant functions to identify shared sgRNAs of a group of genes. Moreover, we also set primer design and sequence extraction functions in CIDP to help users design sgRNAs efficiently.

bioinformatics↗

bioRxiv2022

Response of the organellar and nuclear (post)transcriptomes of Arabidopsis to drought stress

Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used long non-coding RNA-sequencing to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0 and a mutant (protein phosphatase 7-like, pp7l), from which we demonstrated that it can survive long periods of drought stress. The changes detected in the pp7l mutant were marginal, while in the wild type chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Also, transcriptional activation of transposable elements played only a minor role. Conversely, alternative splicing (AS) affected nearly 2,000 genes (11% of expressed nuclear genes). Of these, 25% underwent isoform switching, and 15% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought. Moreover, altering the relative contributions of spliced isoforms might enhance drought resistance. For instance, our data imply that accumulation of a non-functional FLM (FLOWERING LOCUS M) isoform - and not the ratio of functional isoforms as suggested for temperature responses - accounts for the early-flowering phenotype under drought conditions.

plant biology↗

bioRxiv2022

Triangulation fails when neither linguistic, genetic, nor archaeological data support the Transeurasian narrative

Robbeets et al.1 argue that the dispersal of the so-called "Transeurasian" languages, a highly disputed language superfamily comprising the Turkic, Mongolian, Tungusic, Koreanic, and Japonic language families, was driven by Neolithic farmers in the West Liao River region of China. They adduce evidence from linguistics, archaeology, and genetics to support their claim. An admirable feature of the Robbeets et al.s paper is that all their datasets can be accessed. However, a closer investigation of all three types of evidence reveals fundamental problems with each of them. Robbeets et al.s analysis of the linguistic data does not conform to the minimal standards required by traditional scholarship in historical linguistics and contradicts their own stated sound correspondence principles. A reanalysis of the genetic data finds that they do not conclusively support the farming-driven dispersal of Turkic, Mongolian, and Tungusic, nor the two-wave spread of farming to Korea. Their archaeological data contain little phylogenetic signal, and we failed to reproduce the results supporting their core hypotheses about migrations. Given the severe problems we identify in all three parts of the "triangulation" process, we conclude that there is neither conclusive evidence for a Transeurasian language family nor for associating the five different language families with the spread of Neolithic farmers from the West Liao River region.

genetics↗

bioRxiv2022

Uncovering Lasonolide A biosynthesis using genome-resolved metagenomics

Invertebrates, in particular sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anti-cancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60 cell line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed this bacterium, herein named Candidatus Thermopylae lasonolidus, only has 88.78% 16S rRNA identity with the closest relative Pedosphaera parvula Ellin514, indicating it represents a new genus. The lasonolide A (las) biosynthetic gene cluster (BGC) was identified as a trans-AT polyketide synthase (PKS) pathway. When compared with its host genome, the las BGC exhibits a significantly different GC content and penta-nucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed including the presence of three insertions, two single-nucleotide polymorphisms and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the Verrucomicrobial producer shows signs of genome-reduction, its genome size is still fairly large (about 5Mbp) and when compared to its closest free-living relative contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. ImportanceWhile sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small amounts by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations and causes premature chromosome condensation, blebbing, cell contraction and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anti-cancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing to uncover how an uncultured bacterium produces LSA. This provides future opportunities for heterologous expression and cultivation efforts that may minimize LSAs supply problem.

microbiology↗

bioRxiv2022

Activated interstitial macrophages are a predominant target of viral takeover and focus of inflammation in COVID-19 initiation in human lung

Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices cultured ex vivo can be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of "infection pseudotime" for individual lung cell types. This revealed that the prominent SARS-CoV-2 target is a population of activated interstitial macrophages (IMs), which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover of IMs, there is cell-autonomous induction of a pro-fibrotic program (TGFB1, SPP1), and an inflammatory program characterized by the early interferon response, chemokines (CCL2, 7, 8, 13, CXCL10) and cytokines (IL6, IL10), along with destruction of cellular architecture and formation of dense viral genomic RNA bodies revealed by super-resolution microscopy. In contrast, alveolar macrophages (AMs) showed neither viral takeover nor induction of a substantial inflammatory response, although both purified AMs and IMs supported production of infectious virions. Spike-dependent viral entry into AMs was neutralized by blockade of ACE2 or Sialoadhesin/CD169, whereas IM entry was neutralized only by DC-SIGN/CD209 blockade. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated IMs as a prominent site of viral takeover and focus of inflammation and fibrosis, and suggest therapeutic targeting of the DC-SIGN/CD209 entry mechanism to prevent IM infection, destruction and early pathology in COVID-19 pneumonia. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.

genomics↗

bioRxiv2022

Adipokine C1q/Tumor necrosis factor-related protein 3 (CTRP3) Attenuates Intestinal Inflammation via Histone Deacetylase Sirtuin 1 (SIRT1)/NF-κB Signaling

BACKGROUND & AIMSThe adipokine C1q/tumor necrosis factor-related protein 3 (CTRP3) has anti-inflammatory effects in several non-intestinal disorders. Although CTRP3 is reduced in the serum of patients with inflammatory bowel disease (IBD), its function in IBD has not been established. We aimed to elucidate the function of CTRP3 and related molecular mechanisms in intestinal inflammation using a colitis model of genetically-modified CTRP3 mice and intestinal epithelial tissue from patients with Crohns disease (CD), one of the two main forms of IBD. METHODSCTRP3 knockout (KO) and overexpressing transgenic (Tg) mice along with their corresponding wild-type (WT) littermates were subjected to drinking water containing dextran sulfate sodium (DSS) for 6-10 days to induce acute colitis. Mouse colitis symptoms and histological data were analyzed. CTRP3-mediated signaling was examined in the intestinal tissue of mice and patients with CD. RESULTSCTRP3 mRNA and protein were detected in murine and human intestinal epithelial cells, as well as in murine intestinal smooth muscle cells and mesenteric fat. In DSS-induced acute colitis models, CTRP3 KO mice developed more severe colitis than their WT littermates, while CTRP3 overexpressing Tg mice developed less severe colitis than their WT littermates. In both water- and DSS-treated CTRP3 KO mice, reduced CTRP3 levels correlated with decreased levels of Sirtuin 1 (SIRT1), a histone deacetylase, increased levels of phosphorylated nuclear factor kappa B (NF-{kappa}B) subunit p65, resulting in increased expression of pro-inflammatory cytokines tumor necrosis factor- (TNF-) and interleukin 6 (IL-6). The results from CTRP3 Tg mice mirrored those from CTRP3 KO mice in most respects. This CTRP3/SIRT1/NF-{kappa}B relationship was also observed in the intestinal epithelial tissue of patients with active and inactive CD. CONCLUSIONSCTRP3 expression levels correlate negatively with intestinal inflammation in mouse colitis models and CD patients. CTRP3 attenuates intestinal inflammation via SIRT1/NF-{kappa}B signaling to suppress pro-inflammatory cytokines in mouse colitis models and patients with IBD. The manipulation of CTRP3 signaling, including through the use of SIRT1 agonists, may offer translational potential in the treatment of IBD. WHAT YOU NEED TO KNOWO_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSAdipokine C1q/tumor necrosis factor-related protein 3 (CTRP3) is a novel adipokine with known non-intestinal anti-inflammatory effects. CTRP3 is reduced in the serum of patients with inflammatory bowel disease (IBD). However, little is known about whether and how CTRP3 influences intestinal inflammation in IBD. NEW FINDINGSCTRP3 mRNA and protein were detected in murine and human intestinal epithelial cells, as well as in murine intestinal smooth muscle cells and mesenteric fat. CTRP3 deletion was associated with more severe acute dextran sulfate sodium (DSS)-induced colitis, while CTRP3 overexpression was associated with less severe colitis. In both mice and humans, reduced CTRP3 levels correlated with reduced levels of the histone deacetylase Sirtuin 1 (SIRT1), resulting in the up-regulation of phosphorylated nuclear factor-kappa B (NF-{kappa}B) p65 and pro-inflammatory cytokine synthesis. LIMITATIONSThis study was performed using genetically modified mice and human tissue samples. An acute DSS-induced colitis model was used; additional mouse colitis models designed to mimic other aspects of IBD will be examined in future studies. The specific source of the secreted CTRP3 protein which influences intestinal inflammation is yet to be identified. The use of recombinant CTRP3 protein supplementation and SIRT1 agonists to mitigate intestinal inflammation also requires further study. IMPACTCTRP3 is a novel anti-inflammatory adipokine that attenuates intestinal inflammation in colitis mouse models and intestinal epithelial tissue of patients with IBD. CTRP3 attenuates intestinal inflammation by activating SIRT1, which suppresses the pro-inflammatory transcriptional activity of phosphorylated NF-{kappa}B p65. CTRP3 and SIRT1 agonists have potential as novel IBD drug targets.

pathology↗

bioRxiv2022

FASTAptameR 2.0: A Web Tool for Combinatorial Sequence Selections

Combinatorial selections are powerful strategies for identifying biopolymers with specific biological, biomedical, or chemical characteristics. Unfortunately, most available software tools for high-throughput sequencing analysis have high entrance barriers for many users because they require extensive programming expertise. FASTAptameR 2.0 is an R-based reimplementation of FASTAptamer designed to minimize this barrier while maintaining the ability to answer complex sequence-level and population-level questions. This opensource toolkit features a user-friendly web tool, interactive graphics, up to 100x faster clustering, an expanded module set, and an extensive user guide. FASTAptameR 2.0 accepts diverse input polymer types and can be applied to any sequence-encoded selection.

bioinformatics↗

bioRxiv2022

Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade and the role of insertions and extensions in diversification

Ribonucleotide reductases (RNRs) are used by all organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6,779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade O. We employed small-angle X-ray scattering (SAXS), cryogenic-electron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from Synechococcus phage S-CBP4 and report the most minimal RNR architecture to-date. Using the catalytic barrel as a starting point for diversification, we traced the evolutionarily relatedness of insertions and extensions that confer the diversity observed in the RNR family. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study.

bioinformatics↗

bioRxiv2022

The germline factor DDX4 contributes to the chemoresistance of small cell lung cancer cells

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DDX4, a germline factor and RNA helicase conserved in all multicellular organisms, contributes to epithelial mesenchyme transition (EMT)-like features and cisplatin resistance in small cell lung cancer (SCLC) cells. DDX4 depletion in H69AR and SHP77 cell lines decreased motility and resistance to cisplatin, whereas its overexpression increased these features. Proteomic analysis suggests that DDX4 upregulates metabolic protein expression related to DNA repair and immune/inflammatory response, suggesting its fundamental function may be in regulating cellular metabolism. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Although the DDX4 expression level in somatic tumors is generally low compared to that in the germline, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of EMT and cisplatin resistance markers. Taken together, we conclude that DDX4 influences the survival of SCLC patients by altering cellular metabolism in response to environmental cues such as drug treatments. This fundamental function of DDX4 as a germline factor might be applicable in other cancer types that express DDX4 and may serve as a key to combat specific tumors that are highly resistant to treatments. HighlightsO_LIDDX4 contributes to cellular motility and drug resistance in SCLC cells. C_LIO_LIDDX4-overexpression globally alters the proteome and suppresses cytokine production. C_LIO_LIDDX4 promotes tumorigenesis and drug resistance in vitro and in vivo. C_LIO_LIDDX4 expression correlates with survival in SCLC patients and with immune/inflammatory response both in cell lines and patient samples. C_LI

cancer biology↗

bioRxiv2022

Efficient evolution of human antibodies from general protein language models and sequence information alone

Natural evolution must explore a vast landscape of possible sequences for desirable yet rare mutations, suggesting that learning from natural evolutionary strategies could accelerate artificial evolution. Here, we report that deep learning algorithms known as protein language models can evolve human antibodies with high efficiency, despite providing the models with no information about the target antigen, binding specificity, or protein structure, and also requiring no additional task-specific finetuning or supervision. We performed language-model-guided affinity maturation of seven diverse antibodies, screening 20 or fewer variants of each antibody across only two rounds of evolution. Our evolutionary campaigns improved the binding affinities of four clinically relevant antibodies up to 7-fold and three unmatured antibodies up to 160-fold across diverse viral antigens, with many designs also demonstrating improved thermostability and viral neutralization activity. Notably, our algorithm requires only a single wildtype sequence and computes recommended amino acid changes in less than a second. Moreover, the same models that improve antibody binding also guide efficient evolution across diverse protein families and selection pressures, indicating that these results generalize to many natural settings. Contrary to prevailing notions of evolution as difficult and resource-intensive, our results suggest that when constrained to a narrow manifold of evolutionary plausibility, evolution can become much easier, which we refer to as the "efficient manifold hypothesis."

synthetic biology↗