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Feng, L.

Publications and source records attributed to Feng, L..

At least 37 records · Page 2Linked to original sources

Myeloid CD209 impairs T-cell activation by inducing ICAM-2-ERM-dependent cortical stiffening

Resistance to PD-1-based immunotherapy remains a major challenge in cancer treatment, especially in immunologically "cold" tumors characterized by T-cell exclusion and impaired effector function. Tumor-associated myeloid cells are important mediators of this resistance, but whether they directly suppress T-cell immunity through mechanical regulation remains unclear. Here, we identify DC-SIGN (CD209), a myeloid-expressed C-type lectin receptor, as a mechanosuppressive regulator of antitumor T-cell immunity. DC-SIGN is selectively enriched in tumor-infiltrating M2-like macrophages and monocytes, and its expression is associated with poor clinical response to PD-1 blockade and elevated TIDE scores across multiple cancer types. Mechanistically, DC-SIGN directly engages ICAM-2 on T cells and activates ROCK1-ERM signaling. This interaction increases T-cell cortical stiffness, destabilizes T cell-antigen-presenting cell interactions, and attenuates T-cell receptor signaling. Blocking the DC-SIGN-ICAM-2-ERM pathway restores T-cell activation and promotes CD8+ T-cell infiltration and effector function in mouse, humanized, and ex vivo human tumor systems. Importantly, DC-SIGN blockade overcomes resistance to PD-1-based immunotherapy in refractory and immunologically "cold" tumor models, and further enhances the response to PD-1 inhibition. Together, our findings define a myeloid-derived mechanical immune checkpoint and reveal T-cell mechanosuppression as a targetable mechanism of tumor immune evasion and immunotherapy resistance.

immunology↗

MOV10 inhibits SADS-CoV replication by enhancing TRIM24-mediated K63-linked TRAF3 ubiquitination and this inhibition is antagonized by viral N protein

The global outbreak of SARS-CoV-2 has resulted in a renewed focus on coronaviruses with the potential for cross-species transmission and mutation risks. In particular, swine acute diarrhea syndrome coronavirus (SADS-CoV), potentially originating from intermediate horseshoe bat (Rhinolophus affinis), is a novel coronavirus that causes acute diarrhea, vomiting, and high mortality in suckling piglets. Innate immunity plays a crucial role in defending the host against invading pathogens. Previous studies have identified several host factors that inhibit SADS-CoV replication through innate immunity; however, research on SADS-CoV remains insufficient compared to that on other coronaviruses. In this study, the host factor Moloney leukemia virus 10 protein (MOV10) inhibited SADS-CoV replication by promoting interferon (IFN) production. Mechanistically, MOV10 exerted its antiviral effect primarily through its N-terminal domain, which regulated the TNF receptor-associated factor 3 (TRAF3)-mediated innate immune pathway. Specifically, we reveal that MOV10 enhanced tripartite motif-containing 24 (TRIM24)-mediated K63-linked ubiquitination of TRAF3, thereby promoting IFN production and inhibiting SADS-CoV replication. Furthermore, we identified the strategies by which SADS-CoV evaded innate immunity. The viral N protein inhibited the antiviral effect of MOV10 by disrupting interaction between MOV10 and TRAF3, and by promoting K48-linked polyubiquitination of TRAF3, leading to TRAF3 degradation via the ubiquitin-proteasome pathway. Collectively, our findings reveal the role of MOV10 in antiviral immunity and how SADS-CoV evades host immune defense.

molecular biology↗

DIA-based quantitative proteomics reveal the protective effects of quercetin against atopic dermatitis via attenuating inflammation and modulating immune response

Atopic dermatitis is a common inflammatory skin disorder characterised by recurrent eczematous lesions and intense itch. Quercetin is a naturally occurring flavonoid, exhibits antioxidant and anti-inflammatory properties. Previous studies have indicated its beneficial role in managing atopic dermatitis (AD); however, the precise underlying mechanism remains unclear. This study aimed to investigate the protective effects of quercetin against AD and to elucidate its potential mechanisms. We demonstrated that quercetin effectively suppressed the overexpression of pro-inflammatory effector factors--including interleukin (IL)-1{beta}, thymic stromal lymphopoietin (TSLP) and chemokine (C-X-C motif) ligand 1/10/11 (CXCL1/10/11)--in immortalized human keratinocytes (HaCaT) stimulated by TNF-/IFN-{gamma}. Proteomic analysis revealed 88 differentially expressed proteins, suggesting that quercetins therapeutic action involves multiple pathways. Notably, the NOD-like receptor signaling pathway was identified as a key factor. The key proteins related to this pathway including inhibitor of nuclear factor kappa B kinase epsilon (IKBKE), indoleamine 2,3-dioxygenase 1 (IDO1) and chemokine (C-X-C motif) ligand 9 (CXCL9) showed a significant decrease in expression levels after quercetin treatment. Collectively, these results demonstrate that quercetin alleviates AD symptoms by attenuating inflammatory responses, likely through inhibition of the NOD-like receptor signaling pathway.

pharmacology and toxicology↗

Diverse bacterial pattern recognition receptors sense the conserved phage proteome

Recognition of foreign molecules inside cells is critical for immunity across all domains of life. Proteins of the STAND NTPase superfamily1,2, including eukaryotic NOD-like receptors (NLRs), play a central role in this process3,4. In bacteria and archaea, although several STAND families sense phage proteins5-9, their functional diversity remains largely unexplored. We conducted a systematic phylogenetic analysis of prokaryotic STAND NTPases and identified at least 90 structurally distinct families associated with antiviral defense. We first show that the uncharacterized Avs7 family recognizes the major capsid protein (MCP) of tailed phages. Three cryo-EM structures of Salmonella enterica Avs7 reveal an asymmetric, butterfly-shaped tetramer that assembles stepwise via large, MCP-induced conformational changes, incorporating bacterial elongation factor Tu (EF-Tu) as a structural component that enhances defense. Using genetic screens with a library of 687 phage genes, we further show that 13 additional STAND families sense 13 conserved phage proteins, encompassing most of the core structural and replicative components of tailed phages. These include two distinct MCP-sensing families--Avs8 (PD-{lambda}-4) and Avs10 (Erebus/Hypnos/bNACHT64)--and 11 others (Avs11-21) recognizing the portal, portal adaptor, tail nozzle, head-tail connector, tail terminator, tail tube protein, tail assembly chaperone, tape measure protein, DNA polymerase, helicase/RecA-type ATPase, and single-stranded DNA annealing protein, respectively. Together, our findings highlight structurebased pattern recognition and host factor repurposing as fundamental strategies of bacterial immunity.

molecular biology↗

Sampling Function-Related Metastable States of Proteins With DASH

Rational discovery of function-specific protein modulators as well as activity-enhanced engineering proteins underscore the need to identify function-related metastable states (FMSs) of proteins. However, current experimental and computational methods struggle to generate these states directly from their native state (NS), likely because the NS [->] FMS transition is non-spontaneous. To address this challenge, we introduce Deep learning guided Adaptive sampling with seed Selection and Hopping (DASH), integrating both deep learning and physical functions to guide molecular dynamics (MD) simulation towards FMS. DASH successfully sampled NS [->] FMS transitions in 18 cases across two tasks: protein activation and cryptic allosteric site opening. DASH combined with secondary-structure collective variables is further able to sample folding of disordered regions. Compared to existing methods, DASH demonstrates superior performance while requiring comparable simulation time. Crucially, we applied DASH to sample new conformations of proteins, by which revealing new folding states, previously unknown allosteric sites, and potential activators. These predictions have been further verified in wet-lab experiments and crystal structures determinations. Collectively, our framework provides a robust strategy for function-specific pharmacy research and could accelerate future drug discovery efforts.

bioinformatics↗

A division-associated envelope protein, MAB_2363, drives intrinsic resistance and virulence in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to conventional antibiotics, significantly limiting treatment options. Our previous studies established that MAB_2362 (SteA) is a key regulator of cell division that contributes to intrinsic resistance and virulence. Considering that SteA-like proteins often act alongside SteB counterparts, we hypothesized that the adjacent gene MAB_2363 encodes the corresponding SteB-like division regulator. In this study, we found that deletion of MAB_2363 significantly increased susceptibility to multiple antibiotics and disrupted cell wall permeability. Microscopy revealed striking cell division defects in the mutant, including elongated cell morphology and multiple septa. Subcellular localization of a GFP-MAB_2363 fusion protein demonstrated its enrichment at division septa, confirming its direct involvement in cell division. Deletion of MAB_2363 resulted in attenuated virulence, as evidenced by reduced bacterial survival in macrophages and murine infection models. To assess its functional relation with MAB_2362, we compared the single-deletion mutant of MAB_2363 with the single-deletion mutant of MAB_2362 and the double-deletion mutant of MAB_2362-MAB_2363. Notably, the phenotypes of the MAB_2363 mutant, including cell division defects, antibiotic susceptibility, and virulence, were markedly milder than those of the other two mutants. Collectively, these findings indicate that MAB_2363 functions as a secondary but essential division-associated factor that operates during cell division, thereby influencing intrinsic resistance and virulence in M. abscessus.

microbiology↗

Emergence of hypervirulent Klebsiella pneumoniae ST111 with a novel virulence plasmid causing pyogenic liver abscess

Hypervirulent Klebsiella pneumoniae (hvKp)-associated pyogenic liver abscess is typically linked to K1/K2 capsule serotypes and canonical lineages such as ST23, ST65, and ST86. Cases outside these lineages are rare but pose significant public health concerns. We investigated an unusual liver abscess in an immunocompetent patient caused by hvKp sequence type 111 (ST111). Five isolates (KP1-KP5) were recovered, all belonging to the same clonal lineage. Genomic analysis revealed a 181 kb IncHI1B/IncFIB virulence plasmid encoding rmpADC and the salmochelin cluster (iroBCDN), but lacking aerobactin (iuc) and rmpA2 gene loci. Notably, this plasmid also carried the yersiniabactin locus (ybt), which is typically located on the chromosome. Comparative genomics indicated that similar ST111 strains have been reported only in China, suggesting clonal expansion of a previously unrecognized hvKp lineage. Functional assays demonstrated that plasmid curing abolished the hypermucoviscous phenotype, reduced capsule production, and completely attenuated virulence in murine models, confirming the plasmid as the major driver of hypervirulence. Interestingly, plasmid loss enhanced biofilm formation and relieved growth burden, highlighting fitness trade-offs associated with virulence. This study documents a rare case of hvKp ST111 liver abscess and identifies a novel plasmid conferring hypervirulence. Our findings expand the known diversity of hvKp and underscore that non-canonical lineages can acquire potent virulence determinants enabling severe disease. Continued clinical and genomic surveillance is essential to detect emerging hvKp clones before widespread dissemination.

microbiology↗

Deep learning reveals a microprotein atlas in maize and uncovers novel regulators of seed amino acid metabolism

Microproteins represent a vast and functionally important class of genes that remain largely unexplored in plant genomes. Here, we developed DeepMp, a deep learning framework that integrated multi-omics evidence and built the most comprehensive plant microprotein atlas to date, identifying 18,338 high-confidence candidates in maize. The majority of these appear to have originated de novo from regions previously annotated as noncoding, and they show hallmarks of rapid, lineage-specific evolution and pronounced tissue specificity. These novel microproteins were found integrated into core regulatory networks, particularly in organ development and nutrient storage. Focusing on the maize kernel, population-scale analyses linked microprotein expression to natural variation in amino acid content. We functionally validated three grain-filling-specific candidates originating from noncoding regions by CRISPR-Cas9 knockouts, which confirmed their roles as precise modulators of arginine, aspartate, and methionine levels, without pleiotropic effects on kernel morphology. Our study provides a foundational resource and analytical framework, establishes microproteins as a new and functionally important coding layer in maize, and uncovers a previously untapped source of targets for crop improvement.

genomics↗

Proteolytic activation of diverse antiviral defense modules in prokaryotes

Linked protease-effector modules are widespread in prokaryotic antiviral defense, yet the mechanisms of most remain poorly understood. Here we show that four of the most prevalent modules--metallo-{beta}-lactamase (MBL)-fold hydrolase, /{beta}-hydrolase, Pepco, and EACC1--form latent death effectors that are unleashed by site-specific proteolysis. Genetic, biochemical, and structural analyses reveal novel modes of effector licensing. MBL and /{beta}-hydrolase are zymogens activated by cleavage at two distinct sites, and upon proteolysis, MBL becomes a Zn2+-dependent double-stranded DNA nuclease. In contrast, Pepco and EACC1 act as pore-forming toxins via distinct mechanisms: Pepco constitutively oligomerizes into a denaturation-resistant beta barrel that is activated by cleavage after a specific isoleucine in its C-terminal tail, whereas EACC1 monomers assemble into large membrane pores following removal of an autoinhibitory domain. All four modules are fused to diverse sensors to detect a wide range of phage signals, and EACC1-DnaK chaperone fusions suggest a convergence between defense and general stress responses. These findings establish proteolysisgated activation as a dominant, modular logic for anti-phage defense and reveal parallels with eukaryotic innate immunity.

molecular biology↗

pGGTOX: a versatile plasmid platform for recombination-based genome editing across Enterobacteriaceae

Precise genome editing in Enterobacteriaceae is essential for studying gene function, pathogenesis, and antimicrobial resistance, yet many current systems face host-specific and efficiency limitations. We developed pGGTOX, a modular plasmid platform that enables efficient homologous recombination-mediated genome editing across diverse Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Enterobacter intestinihominis. The system integrates a rhamnose-inducible toxin (MqsR) for stringent counterselection, a sfGFP reporter for visual tracking of recombination events, Golden Gate cloning for rapid assembly of homologous arms, an FRT-flanked resistance cassette for marker removal, and an oriT sequence for conjugative transfer. Together with the companion plasmid pCP20-oriT, pGGTOX supports precise, marker-free genomic modification. Using pGGTOX, we achieved targeted deletions of dapA in E. coli and mrkCD in carbapenem-resistant K. pneumoniae, both with 100% efficiency. The dapA mutant exhibited diaminopimelate auxotrophy, while mrkCD deletion markedly reduced biofilm formation, consistent with the loss of function associated with these genes. pGGTOX also enabled deletion of a 43.1-kb type IV secretion gene cluster (tra) from an IncN/FII plasmid in E. intestinihominis and insertion of a 10-kb CRISPR-Cas9 plasmid-curing module (pCasCure) into an S. enterica IncX1 plasmid. Deletion of the tra gene cluster resulted in a substantial reduction in plasmid conjugation efficiency. Conjugative transfer of the engineered IncX1-pCasCure plasmid into K. pneumoniae facilitated CRISPR-mediated curing of blaKPC, sensitizing carbapenem resistance to susceptibility. In summary, pGGTOX provides a versatile, efficient, and broadly applicable platform for genome engineering and CRISPR delivery in Enterobacteriaceae, expanding the toolkit for bacterial genetics and translational antimicrobial research. ImportancePrecise genetic manipulation in Enterobacteriaceae remains a major technical challenge, particularly for non-model or multidrug-resistant strains. We developed pGGTOX, a versatile and broadly applicable plasmid platform that enables efficient, marker-free genome editing through homologous recombination. By integrating stringent counterselection, visual screening, modular cloning, and conjugative transfer, pGGTOX simplifies construction and streamlines editing across multiple clinically relevant species. We demonstrate its utility in deleting chromosomal and plasmid-borne loci, inserting large genetic modules, and delivering CRISPR-Cas9 systems for targeted elimination of antibiotic resistance genes. This platform expands the molecular toolkit for functional genomics and provides a powerful new strategy for dissecting bacterial virulence, resistance, and plasmid biology.

microbiology↗

Distinct and Shared Impacts of Virulence Plasmids on the Phenotype and Transcriptome in Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae

The global rise of convergent carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) represents a major clinical challenge, yet the role of virulence plasmids (pVirs) in shaping bacterial physiology and pathogenicity remains incompletely understood. Using a CRISPR-Cas9-based curing system, we precisely eliminated pVirs from two clinical CR-hvKp strains with distinct genetic backgrounds, ST23-KL1 (blaNDM-1) and ST11-KL64 (blaKPC-2). Loss of pVir conferred fitness advantages in vitro, reduced capsule production and hypermucoviscosity, and promoted biofilm formation, while markedly attenuating virulence in murine sepsis models. Despite this reduction, the pVir-cured ST23-KL1 strain retained higher virulence than the pVir-cured ST11-KL64 strain, underscoring the contribution of chromosomal background to pathogenic potential. Transcriptomic profiling revealed both shared and strain-specific transcriptional responses to pVir deletion, with broader perturbations observed in the ST11-KL64 strain. pVir removal had limited effects on antibiotic MICs. Complementation experiments further demonstrated differential regulatory roles of the rmpADC and rmpA2D2 operons in capsule expression and hypermucoviscosity across the two strains. Together, these findings establish pVirs as central determinants of CR-hvKp virulence and highlight complex host-plasmid interactions that influence bacterial adaptation and pathogenicity. IMPORTANCEThe emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) poses a critical threat to global health, yet the contribution of virulence plasmids (pVirs) to bacterial fitness and pathogenicity remains poorly defined. By employing a CRISPR-Cas9-based curing strategy, we dissected the role of pVirs in two genetically distinct CR-hvKp strains and uncovered their multifaceted impact on capsule production, hypermucoviscosity, biofilm formation, and virulence. Our findings reveal that pVir loss confers fitness advantages in vitro while attenuating virulence in vivo, with strain-specific transcriptional responses and differential regulation by rmp operons. These results underscore the complex interplay between plasmid-encoded and chromosomal determinants in shaping CR-hvKp pathogenicity and adaptation, offering mechanistic insights that may inform future therapeutic strategies targeting plasmid-mediated virulence.

microbiology↗

Reprogrammable bacterial nanosyringes for RNA and gene editors delivery

The engineered Photorhabdus virulence cassette (PVC) has not yet achieved the RNA packaging and injection delivery, although it can accurately deliver diverse proteins. Fortunately, RNA-binding proteins have a high affinity for binding to specific sequences and are excellent anchoring components between RNA and the inner tube of PVC. By combining the U1A RNA-binding domain (U1A RBD) and PVC, we developed a convenient, RNA non-degradable, and universal PVC-based RNA delivery tool, establishing DART (PVC Docker-based All-purpose RNA Injection Delivery Tool). DART successfully achieved the delivery of diverse RNAs, including Pepper RNA, gRNA, siRNA, and miRNA. We also demonstrated that the DART enabled effective knockout of GFP in vivo and in vitro. Our DART system provided a powerful platform for RNA and gene editing therapeutics in vivo.

molecular biology↗

Expression screening and functional verification of recombinant hirudin from novel chassis cell Chlamydomonas reinhardtii

Hirudin is a potent anticoagulant peptide whose clinical application is limited by scarce natural supplies and the suboptimal activity of recombinant versions from conventional microbial hosts lacking key post-translational modifications. Here, for the first time, we establish the "Generally Regarded as Safe" (GRAS) microalga, Chlamydomonas reinhardtii, as a novel biological chassis for producing fully functional hirudin. Using high-density heterotrophic fermentation, we generated a bioactive lyophilized algal powder suitable for oral delivery. Crucially, the algal-derived hirudin underwent proper tyrosine sulfation--a key modification absent in prokaryotic hosts--conferring exceptionally high thrombin-inhibitory activity (up to 20,000 ATU/mg). When orally administered to a murine thrombosis model, this hirudin-loaded alga demonstrated potent antithrombotic efficacy. It also exhibited a superior safety profile, showing no signs of the thrombocytopenia or bleeding associated with traditional anticoagulants. This study establishes a green, scalable biomanufacturing and oral delivery platform for therapeutics, highlighting transformative potential at the nexus of agricultural bioengineering, functional foods, and biomedicine.

synthetic biology↗

A Rare Multipotent Peg-like Epithelial Cell is a Candidate Cell-of-Origin for High-Grade Serous Ovarian Cancer

To illuminate the origins of high-grade serous ovarian cancer (HGSOC), the most lethal and common form of ovarian cancer, we have created a comprehensive living organoid biobank of human fallopian tube tissue, which is thought to be the origin of this cancer. Through optimized culture protocols and integrated multi-omic profiling--including single-cell RNA sequencing, chromatin accessibility (ATAC) analysis, proteomics, and secretomics--we assembled the largest molecular atlas of the fallopian tube epithelium to date. This resource revealed diverse epithelial lineages and regulatory networks, including a rare, multipotent epithelial subpopulation with hybrid epithelial-mesenchymal features. Spatially localized to the basal epithelium and resembling mesonephric developmental precursors, these cells exhibit transcriptomic and proteomic similarities to the mesenchyme-like subtype of HGSOC, implicating them as potential cells-of-origin. Their molecular identity is preserved in organoid models, enabling future mechanistic and translational studies. This resource, which advances fundamental understanding of epithelial hierarchy and cancer susceptibility, provides a platform to inform early detection and prevention strategies for aggressive forms of ovarian cancer. HighlightsO_LIEstablishment of a clinically annotated fallopian tube organoid biobank enables delineation of epithelial lineage hierarchies and differentiation capacity. C_LIO_LIMulti-omics integration defines robust, lineage-specific transcriptional and regulatory networks in the fallopian tube epithelium. C_LIO_LIA rare basal epithelial subpopulation with mesenchymal features aligns with a mesenchyme-like subtype of high-grade serous ovarian cancer. C_LIO_LIRare basal peg cells exhibit fetal mesonephric developmental transcriptional programs and are maintained ex-vivo in fallopian tube organoids. C_LI

cancer biology↗

A Toolkit for In Vivo Mapping and Modulating Neurotransmission at Single-Cell Resolution

Understanding the organization and regulation of neurotransmission at the level of individual neurons and synapses requires tools that can track and manipulate transmitter-specific vesicles in vivo. Here, we present SynaptoTagMe, a suite of genetic tools in Caenorhabditis elegans to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the C. elegans nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.

neuroscience↗

Super-pangenome analysis of 3562 human and animal papillomavirus isolates illuminates their genome and pathogenicity evolution

Papillomaviruses (PVs) are groups of ubiquitous DNA viruses infecting a wide range of vertebrate hosts, including human. The infection of some human PVs (i.e., HPVs) are strongly associated with cancers, but it remains elusive how such pathogenicity evolved at the genomic level. Here we performed a super-pangenome analysis on a compendium of well-curated 3562 human and animal PV genomes. We found their global genomic diversity being strongly associated with their body sites and tissue origins, but not with their host, geographical, or disease specificity. By reconciling time-resolved phylogenies of PVs and their hosts, we revealed a high-resolution view on how virus-host cospeciation, horizontal host switches, and duplication/loss turnovers collectively shaped the evolution of PVs. We identified key selection-driven mutations that defined the genus-specific divergence of PVs, with those underlying the rise of Alpha-PVs highly enriched in the N-terminal domain of E1 gene. Especially, we identified a group of risk-defining variant sites showing contrasting genotypes between low-risk and high-risk HPVs. With four independent PV genome cohorts, we further validated their high sensitivity and specificity on HPV risk classification even at single site level, underscoring their application values in large-scale HPV screening tests. Finally, with cervical and oropharyngeal cancer patient cohorts assembled by this study, we also evaluated the prognostic value of specific HPV16 variants in predicting patient survival. Taken together, such illumination on the genome and pathogenicity evolution of PVs paves the road for better prevention, control, and treatment of PV-related cancers. Significance statementPapillomaviruses (PVs) are ancient and widespread DNA viruses co-evolved with many vertebrate hosts, including human. Some human PVs (e.g., HPV16 and HPV18) bear strong association with cancers, raising a pressing public health challenge. A better understanding of how such pathogenicity evolved holds the key in addressing this challenge. Here we systematically investigated the genomic and evolutionary diversity of PVs via a pangenome study on over 3500 human and animal PV isolates, which revealed key selection-driven mutations shaping their genome and pathogenicity evolution. With independently acquired HPV-associated cancer cohorts, we further demonstrated how some of these important HPV variants can serve as clinically informative predictive and prognostic biomarkers. Such comprehensive characterization on PVs genome diversity, pathogenicity evolution, and clinical implication paves the road for better prevention, control, and treatment of PV-related cancers.

genomics↗

A conserved small RNA-generating gene cluster undergoes sequence diversification and contributes to plant immunity

Small RNA-mediated gene silencing contributes to plant immunity. The secondary small interfering RNA (siRNA) pathway promotes defense by silencing target genes in invading fungal and oomycete pathogens. Many secondary siRNAs derive from transcripts potentially encoding pentatricopeptide repeat (PPR) proteins. Here, we report that siRNA production is an ancient function of an evolutionarily conserved clade of PPR genes that undergo extensive within-species diversification. In Arabidopsis thaliana, siRNA-source PPRs are physically clustered in one locus on Chromosome 1. These sequences are diversified through gene duplication followed by sequence diversification as well as accumulation of high-impact variations including pseudogenization. This diversity leads to the accumulation of a diverse PPR-siRNA pool, consistent with an engagement in a co-evolutionary arms race with the pathogens. This study defines siRNA-producing PPRs as a family of defense genes and highlights the potential of PPR-siRNA-based engineering for enhancing broad-spectrum disease resistance.

plant biology↗

Comparison of the 2-CT method and the 2-ΔΔCT method for real-time qPCR data analysis

[purpose]The [Formula] method proposed by Livak and Schmittgen in 2001 is used for real-time quantitative polymerase chain reaction (RT-qPCR) data analysis. This methods fundamental logic involves normalizing all data against the control group to achieve relative quantification of gene expression. During our practical calculations, we identified an inherent bias in this method: the practice of directly taking arithmetic means of a set of C (or {Delta}C) values during [Formula] calculations fails to fully account for the exponential nature of CT values (where CT values serve as exponents in power-of-2 operations), leading to deviations in computational results. In this study, we elucidate this systematic bias and propose an innovative approach to circumvent it. [Methods]We propose the [Formula] method, which bases calculations on [Formula] values and computes the fold-change of target genes relative to control group target genes. The computational logic of the [Formula] method strictly adheres to the exponential characteristics of CT values, avoiding the use of arithmetic averaging at CT and {Delta}CT levels, thereby more accurately reflecting gene expression levels in datasets. In this paper, we detail the computational process of the [Formula] method and compare results from both methodologies. [Results]Calculations based on Livak and Schmittgens published data show differences between the two methods, though the discrepancies are relatively small. In calculations from our recent cadmium exposure experiments, the [Formula] method indicates that 8-hour cadmium exposure increases irg-6 gene expression in C. elegans from 1.314 to 7.125-fold, while the [Formula] method shows an increase from 1 to 4.124-fold. The two methods exhibit nearly 70% discrepancy in irg-6 gene fold-change quantification, with statistical p-values of 0.0002 and 0.0015 respectively. In all calculations presented in the paper, the [Formula] method fails to produce an exact unit value (mean fold-change = 1) for control groups, whereas the [Formula] method consistently yields control group results of exactly 1, demonstrating its capability for precise normalization. [Conclusions]In summary, the [Formula] method demonstrates superior computational rigor, and we recommend its adoption in RT-qPCR data analysis. This improvement proves particularly valuable for experimental datasets with substantial CT value variability among different samples, establishing a more reliable computational paradigm for RT-qPCR analysis.

molecular biology↗