Results 751-775 of about 1,000
  1. Soybean transformation remains challenging and has not kept pace with the rapid advancement of genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 ( WUS2 ) and isopentenyltransferase ( IPT ) achieved higher transformation efficiencies (15.2% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produces heritable transgenic events within 9-11 weeks and successfully delivers CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2 / IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Together, our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation efficiency, reduces tissue culture requirements, and offers a promising genome editing platform for soybean improvement.
    Date: 2025-03-07
    Authors: Alok A, Raman V, D’Agostino L, Kshetry AO, Rai KM, Wang C, Gunapati S, Stupar RM, Patil GB, Zhang F.
    Ref: bioRxiv
  2. Background Calcific aortic valve stenosis (AS) affects 3% of older adults and lacks medical treatment. The deacetylase Sirtuin 1 (SIRT1) could be involved in many pathways linked to AS progression. Sodium-glucose co-transporter 2 inhibitors (SGLT2i), glucose-lowering agents, have been shown to reduce cardiovascular events (likely via SIRT1), but their possible benefits in AS are unknown. Our study aims to uncover the role of SIRT1 in AS progression and assess the benefit of SGLT2i to slow down the aortic valve fibro-calcification processes. Methods RNA-seq data of human aortic valve specimens were collected from ARChS4 database. SIRT1 knockdown (SIRT1 KD) and overexpressing (SIRT1 Over) valve interstitial cells (VIC) were generated by CRISPR/Cas9. Real-time PCR, immunofluorescence, and calcification assays were used to characterized mutant VICs. Conditioned medium experiments were implemented to evaluate SGLT2i effect on cellular cross-talk and calcification. Diabetic patients’ data from the Lombardy regional healthcare database, treated with sulphonylureas (SU; no effect on SIRT1) and SGLT2i (acting on SIRT1), were selected and matched 1:1 by age, sex, and multisource comorbidity score. Cumulative incidence of hospitalization for non-rheumatic aortic valve disease was assessed by Kaplan-Meier and multivariable Cox proportional hazards models were used to estimate hazard ratios. Results RNA-seq showed that SIRT1 could be a master regulator of multiple AS-related pathways. Functional studies on mutant VICs revealed that SIRT1 directly regulates antioxidant processes, extracellular-matrix remodeling and calcification by modulating key transcription factors. Moreover, calcification assays further support this role, revealing an increased calcification in SIRT1 KD VICs and a concomitant decrease in VIC SIRT1 Over when compared to wild type. Then, exploring SGLT2i impact on calcification, we showed that VICs cultured in SGLT2i-treated-endothelial medium exhibited reduced calcification associated with endothelial-increased nitric oxide levels, while SIRT1 inhibition enhanced VIC calcification. The real-world data analysis revealed that SGLT2i-treated group had a lower incidence of hospitalized patients for non-rheumatic aortic valve disease compared to SU-treated group. Conclusions Our data identify SIRT1 as a key regulator of fibro-calcific processes in AS and suggest that SGLT2i may slow the aortic valve degeneration through SIRT1 modulation. These findings highlight SGLT2i as a promising therapeutic option for AS prevention and care. Clinical Perspectives What is new? Sirtuin 1 (SIRT1) downregulation is linked to the progression of aortic stenosis (AS) pathological processes and plays a crucial role in mitigating oxidative stress, fibrosis, and calcification. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) treatment of valve endothelial cells results in the secretion of protective factors that reduce valve interstitial cell calcification, highlighting the valuable role of endothelial health in preventing valve degeneration. Real-world data indicate that the use of SGLT2i is associated with a lower incidence of hospitalization rate for aortic valve disease as compared to sulfonylureas, suggesting a protective effect against AS in diabetic patients. What are the clinical implications? This study highlights the potential of restoring SIRT1 activity as a therapeutic strategy to mitigate pro-calcific and pro-fibrotic processes in AS. SGLT2i may offer a breakthrough therapeutic option for AS, a condition currently lacking effective pharmacological treatments, providing new hope for slowing disease progression and improving clinical outcomes.
    Date: 2025-03-06
    Authors: Valerio V, Massaiu I, Franchi M, Rimbert A, Begué-Racapé Z, Chiesa M, Myasoedova VA, Rusconi V, Bertolini F, De Giorgi D, Bonomi A, Pirola S, Zanobini M, Di Pietro P, Carrizzo A, Ciccarelli M, Capoulade R, Genovese S, Poggio P.
    Ref: bioRxiv
  3. 1 Characterization of essential genes across the genome is fundamental to understanding cellular functions at a molecular level. While significant progress has been made in characterizing essential genes in human and mouse models, relatively little is known about essential genes in the porcine genome. Pigs are an important production species and are now emerging as valuable models for studying human diseases due to their physiological similarities to humans. To map essential genes across the porcine genome, we have developed a novel porcine genome-wide CRISPR knockout screening library (pGeCKO) and applied it to two porcine cell lines, PK15 and IPEC-J2. We identified 2,245 essential genes in PK15 cells and 919 essential genes in IPEC-J2 cells, with 683 of these shared between both cell lines. Functional analyses revealed that most essential genes are involved in core cellular processes such as cell cycle regulation, DNA replication, transcription, and translation. Comparative analysis with human essential genes from the DepMap project revealed that over half of the genes are shared with humans and the rest are porcine-specific. These porcine-specific essential genes included genes in core functional pathways related to protein and RNA processing as well as many related to N-glycan biosynthesis, signal transduction, and several long-noncoding RNAs. This work provides a new resource for leveraging porcine models in disease research, enhancing our understanding of porcine genetics and its implications for human health.
    Date: 2025-03-06
    Authors: Harvey T, Boyartchuk V, van Son M, Gjuvsland AB, Grindflek E, Kent M.
    Ref: bioRxiv
  4. PML nuclear bodies (PML-NBs) are dynamic subnuclear structures important for chromatin dynamics and anti-viral defense. In this study we investigate the role of Sp100 isoforms in promoting localization of the H3.3 histone chaperone HIRA to PML-NBs in human keratinocytes. Sp100 knockout (KO) cell lines were generated using CRISPR-Cas9 technology and shown to display normal keratinocyte differentiation and PML-NB formation. However, HIRA and its associated complex members (UBN1 and ASF1a) failed to localize to PML-NBs in the absence of Sp100, even after interferon stimulation. Exogenous expression of the four main isoforms of Sp100 showed that the Sp100A isoform is the primary driver of HIRA localization to PML-NBs, with the SUMO interacting motif (SIM) playing an important role. These findings highlight the functional diversity of the Sp100 isoforms in modulating chromatin dynamics at PML-NBs.
    Date: 2025-03-06
    Authors: Della Fera AN, Arman W, Powers ME, Warburton A, McBride AA.
    Ref: bioRxiv
  5. ABSTRACT Serine incorporator 5 (SERINC5) is a host restriction factor that targets certain enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). It integrates into the viral envelope from the cell surface, inhibiting viral entry. SERINC5 is transported to the cell surface via polyubiquitination, while a single K130R mutation retains it in the cytoplasm. Both HIV-1 Nef and MLV glycoGag proteins antagonize SERINC5 by reducing its expression in producer cells. Here, we report that MLV glycoGag employs selective autophagy to downregulate SERINC5, demonstrating a more potent mechanism for decreasing its cell surface expression. Although glycoGag is a type II integral membrane protein, it primarily localizes to the cytoplasm and undergoes rapid proteasomal degradation. Employing the K130R mutant, we show that Nef, primarily associated with the plasma membrane, downregulates SERINC5 only after it has trafficked to the cell surface, whereas glycoGag can reduce its expression before reaching the plasma membrane while still in the cytoplasm. Nonetheless, an interaction with SERINC5 stabilizes and recruits glycoGag to the plasma membrane, enabling it to downregulate SERINC5 from the cell surface. Through affinity-purified mass spectrometry analysis combined with CRISPR/Cas9 knockouts, we find that glycoGag’s activity depends on reticulophagy regulator 1 (RETREG1), an ER-phagy receptor. Further knockout experiments of critical autophagy genes demonstrate that glycoGag downregulates cytoplasmic SERINC5 via micro-ER-phagy. These findings provide crucial new insights into the ongoing arms race between retroviruses and SERINC5 during infection. AUTHOR SUMMARY HIV-1 Nef and MLV glycoGag are unrelated viral proteins, yet both counteract the same host restriction factor, SERINC5, to facilitate productive infection. In this study, we report a novel pathway through which glycoGag downregulates SERINC5. We demonstrate that while Nef downregulates SERINC5 only after it has trafficked to the cell surface, glycoGag can directly downregulate SERINC5 in the cytoplasm before it reaches the plasma membrane. Furthermore, we show that this pathway is mediated by the ER-phagy receptor RETREG1, which targets SERINC5 for degradation via micro-ER-phagy. This mechanism provides a more effective means of blocking SERINC5 antiviral activity. These findings reveal that retroviruses have evolved different strategies to antagonize SERINC5, highlighting the critical role of SERINC5 in restricting retroviral infections.
    Date: 2025-03-06
    Authors: Ahmad I, Zhang J, Li R, Su W, Liu W, Wu Y, Khan I, Liu X, Li L, Li S, Zheng Y.
    Ref: bioRxiv
  6. ABSTRACT Producing the gene-knockout mutant is a critical strategy in reverse genetics for gene functional analyses. Plant science has applied various mutagenesis, including chemical mutagen, T-DNA insertion, and genome editing. The intact gene expression is completely disrupted, while the mutant retains the intact sequence region. Lately, effective whole open reading frame (ORF) deletion through the CRISPR/Cas9 system using multiplex guide RNAs was reported in Arabidopsis, targeting a gene expressed in somatic cells. Here we applied the scheme targeting the reproductive genes GENERATIVE CELL SPECIFIC 1 ( GCS1 ), GAMETE EXPRESSED 2 ( GEX2 ), DUF679 DOMAIN MEMBRANE PROTEIN 8 ( DMP8 ), and DMP9 , which are fertilization regulators. Homozygous gene deletion lines were successfully obtained in the T1 generation, with acquisition rates ranging from 8.3% to 30.0%. The rates approximately correlated with the scores predicted by the DeepSpCas9. The analysis of the GCS1 deletion line ( Δgcs1 ) suggested that avoiding the first bolt removal is important for consistent phenotype of the developed inflorescences. Compared to the previously reported mutants, the GEX2 deletion line ( Δgex2 ) showed a difference in seed development phenotype, indicating that the remaining intact gene region in the mutant could unexpectedly influence the function. Simultaneous deletions of DMP8 and DMP9 , which are in distinct chromosomes, were also succeeded in the T1 generation. The obtained results showed that all deletions were inheritable. The scheme challenged here demonstrated the effective production of homozygous mutants for the genes, even if the reproductive lethal or recessive.
    Date: 2025-03-06
    Authors: Yoshimura A, Seo Y, Kobayashi S, Igawa T.
    Ref: bioRxiv
  7. ABSTRACT The phenotype of a mutation often differs across genetically distinct individuals. In the most extreme case, a gene can be essential for viability in one genetic background, but dispensable in another. Although genetic context-dependency of mutant phenotypes is frequently observed, the underlying causes often remain elusive. Here, we investigated the genetic changes responsible for differences in gene essentiality across 18 genetically diverse natural yeast strains. First, we identified 39 genes that were essential in the laboratory reference strain but not required for viability in at least one other genetic background, suggesting that the natural strain contained suppressor variants that could bypass the need for the essential gene. We then mapped and validated the causal bypass suppressor variants using bulk segregant analysis and allele replacements. Bypass suppression was generally driven by a single modifier gene that tended to differ between genetic backgrounds. The suppressors often indirectly counteracted the effect of deleting the essential gene, for instance by changing the transcriptome of a cell. Context-dependent essential genes and their bypass suppressors were frequently co-mutated across 1,011 yeast isolates and identified naturally occurring evolutionary trajectories. Overall, our results highlight the relatively high frequency of bypass suppression in natural populations, as well as the underlying variants and mechanisms. A thorough understanding of the causes of genetic background effects is crucial for the interpretation of genotype-to-phenotype relationships, including those associated with human disease.
    Date: 2025-03-06
    Authors: Batté A, Bosch-Guiteras N, Pons C, Ota M, Lopes M, Sharma S, Tellini N, Paltenghi C, Conti M, Kan KT, Ho UL, Wiederkehr M, Barraud J, Ashe M, Aloy P, Liti G, Chabes A, Parts L, van Leeuwen J.
    Ref: bioRxiv
  8. Understanding how genetic variants drive phenotypic differences is a major challenge in molecular biology. Single nucleotide polymorphisms form the vast majority of genetic variation and play critical roles in complex, polygenic phenotypes, yet their functional impact is poorly understood from traditional gene-level analyses. In-depth knowledge on the impact of single nucleotide polymorphisms has broad applications in health and disease, population genomic or evolution studies. The wealth of genomic data and available functional genetic tools make Drosophila melanogaster an ideal model species for studies at single nucleotide resolution. However, to leverage these resources and potentially combine it with the power of functional genetics to enhance genotype-phenotype research, it is essential to develop techniques to predict functional impact and causality of single nucleotide variants. Here, we present FlyCADD, a functional impact prediction tool for single nucleotide variants in D. melanogaster . FlyCADD, based on the Combined Annotation-Dependent Depletion (CADD) framework, integrates over 650 genomic features - including conservation scores, GC content, and DNA secondary structure - into a single metric reflecting a variant’s predicted impact on evolutionary fitness. FlyCADD provides impact prediction scores for any single nucleotide variant on the D. melanogaster genome. We demonstrate FlyCADD’s utility with some examples of application, including the ranking of phenotype-associated variants to prioritize variants for follow-up studies, evaluation of naturally occurring polymorphisms, and refining of CRISPR-Cas9 experimental design. FlyCADD provides a powerful framework for interpreting impact of any single nucleotide variant in D. melanogaster , thereby improving our understanding of genotype-phenotype connections. Article summary Single nucleotide polymorphisms (SNPs), the most common form of genomic variation, play key roles in micro-evolution and adaptation. In Drosophila melanogaster , many SNPs have been associated to phenotypes through association studies, yet functional validation remains challenging and experimental evidence for functional impact is rare. Here, we present FlyCADD, an impact prediction tool that integrates high-quality D. melanogaster genome annotations into a single score reflecting predicted impact of a SNP. FlyCADD can be applied to distinguish causal from neutral variants, for variant prioritization prior to functional studies, and to enhance interpretation of natural variation, thereby improving our understanding of genotype-phenotype relationships.
    Date: 2025-03-06
    Authors: Beets J, Höglund J, Kim BY, Ellers J, Hoedjes KM, Bosse M.
    Ref: bioRxiv
  9. A temperate N-15-like phage and an extensively drug-resistant (XDR) Klebsiella pneumoniae strain were studied in this research. The former was found in hospital wastewater, while the latter was retrieved from the sputum of an intensive care unit patient. The bacteria showed strong resistance to several antibiotics, including penicillin (≥16 μg/mL), ceftriaxone (≥32 μg/mL), and meropenem (≥8 μg/mL), which was caused by SHV-11 beta-lactamase, NDM-1 carbapenemase, and porin mutations (OmpK37, MdtQ). Yersiniabactin, enterobactin, and E. coli common pilus (ECP) genes were also present in the genome; these genes are essential for the acquisition of iron, adhesion, and immune evasion, among other virulence factors. kappa testing categorized the strain as K64 and O2a types. The presence of colicin genes, IncHI1B_1_pNDM-MAR and IncFIB replicons, and other plasmids in this strain demonstrate its ability to spread antibiotic resistance and facilitate horizontal gene transfer. Adding to its genetic variety and adaptability, the genome included CRISPR-Cas systems and eleven prophage regions. The 172,025 bp linear genome and 46.3% GC content of the N-15-like phage showed strong genomic similarities to phages of the Sugarlandvirus genus, especially those that infect K. pneumoniae. There were structural proteins (11.8 percent of ORFs), DNA replication and repair enzymes (9.3 percent of ORFs), and a toxin-antitoxin system (0.4 percent of ORFs) encoded by the phage genome. A protelomerase and ParA/B partitioning proteins indicate that the phage is replicating and maintaining itself in a manner similar to the N15 phage, which is renowned for maintaining a linear plasmid prophage throughout lysogeny. Lysogeny and horizontal gene transfer are two mechanisms by which phages may influence bacterial evolution. Learning about the phage’s role in bacterial evolution, host-phage relationships, and horizontal gene transfer is a great benefit. Understanding the dynamics of antibiotic resistance and pathogen development requires knowledge of phages like this one, which are known for their temperate nature and their function in altering bacterial virulence and resistance profiles. The regulatory and structural proteins of the phage also provide a model for research into the biology of temperate phages and their effects on microbial communities. The importance of temperate phages in bacterial genomes and their function in the larger framework of microbial ecology and evolution is emphasized in this research. Author Summary Antibiotic-resistant bacteria represent a significant global health threat, and comprehending their interactions with bacteriophages is essential for formulating novel antimicrobial tactics and elucidating the molecular development of bacteria. This work examined an extensively drug-resistant (XDR) strain of Klebsiella pneumoniae isolated from an ICU patient and a temperate N-15-like phage identified in hospital effluent. The bacterial strain exhibited resistance to multiple antibiotics owing to an array of resistance genes, plasmids, porin mutations, and virulence characteristics that facilitated its survival and pathogenicity. The genomic investigation of the phage elucidated its structural organization, replication mechanisms, and possible contribution to bacterial development through lysogeny and horizontal gene transfer. Our findings underscore the intricate host-phage interactions that affect antibiotic resistance and pathogenicity in K. pneumoniae, offering significant insights into microbial evolution and the prospective relevance of phages in therapeutic approaches.
    Date: 2025-03-06
    Authors: Yahya R, Albaqami A, Alzahrani A, Althubiti S, Alhariri M, Alrashidi ET, Alhazmi N, Al-Matary MA, Alharbi N.
    Ref: bioRxiv
  10. Abstract Various cellular stresses release immunogenic molecules and elicit immunosurveillance to detect and eliminate potential threats, thus safeguarding organismal health. Nucleolar stress is characterized by the disruption of nucleolar morphology and function, leading to impaired ribosome biogenesis and activation of stress response pathways. However, the regulatory mechanisms linking nucleolar stress to tumour-intrinsic immunogenicity and its therapeutic implications remain unclear. Utilizing CRISPR screens to identify cancer-specific immunogenic regulators, we identified nucleolar OTUD4 (an ovarian tumour domain-containing deubiquitinase) as a repressor of immunogenicity by preserving nucleolar homeostasis. OTUD4 depletion induced nucleolar stress, thereby enhancing immunogenicity and retarding tumor growth in preclinical models. Mechanistically, OTUD4 functioned as a phospho-activated K63 deubiquitinase, catalyzing the K63-linked deubiquitination of nucleophosmin 1 (NPM1) to stabilize its oligomerization. Consequently, OTUD4 deletion promoted NPM1 hyperubiquitination and depolymerization, thereby driving the release of NPM1-binding heterochromatin H3K9me3. The heterochromatin remodeling derepressed endogenous retroelements (ERV) that subsequently stimulated cytosolic DNA-sensing pathways to trigger the type I interferon response and upregulate the expression of antigen presentation genes. Concurrently, ERV-encoded retroviral antigens augmented tumour-intrinsic antigenicity. A pharmacological screen revealed that the casein kinase II inhibitor CX-4945 phenocopied OTUD4 ablation by dephosphorylating and inactivating OTUD4 activity. Clinically, OTUD4 amplification or a high OTUD4-nucleolus gene signature inversely correlated with patient survival and immunotherapy resistance. Our findings position nucleolar stress as a central driver of tumour-intrinsic immunogenicity, and propose a clinical rationale for leveraging nucleolar stress inducers to sensitize immunologically cold tumours to immunotherapy.
    Date: 2025-03-06
    Authors: Wang P, Wu Q, Gao Z, Pei W, Wang P, Yu X, Liu Z, He R, Tian E, Yang Y, Li B, Niu T, Liu Y, Kepp O, Kroemer G, FANG L.
    Ref: Research Square
  11. Antimicrobial resistance (AMR) threatens global health. In this manuscript, I review recent literature underscoring the promise of engineered bacteriophages and CRISPR-Cas systems as targeted strategies against resistant bacteria. These approaches offer alternatives to broad-spectrum antibiotics by precisely disrupting biofilms and inactivating resistance genes—whether applied independently or in tandem. I also underscore the essential role of public-private partnerships in advancing clinical applications and catalyzing the translation of innovative research into practice
    Date: 2025-03-05
    Authors: Dillac L.
    Ref: Preprints.org
  12. CD19-CAR-T-cells emerge as a major therapeutic option for relapsed/refractory B-cell-derived malignancies, however approximately half of patients eventually relapse. To identify resistance-driving factors, we repeatedly exposed B-cell lymphoma/B-cell acute lymphoblastic leukemia to 4-1BB/CD28-based CD19-CAR-T-cells in vitro . Generated models revealed costimulatory domain-dependent differences in CD19 loss. While CD19-4-1BB-CAR-T-cells induced combination epitope/total CD19 protein loss, CD19-CD28-CAR-T-cells did not drive antigen-escape. Consistent with observations in patients relapsing after CD19-4-1BB-CAR-T-cells, we identified CD19 frameshift/missense mutations affecting residues critical for FMC63 epitope recognition. Mathematical simulations revealed that differences between CD19-4-1BB- and CD19-CD28-CAR-T-cells activity against low-antigen-expressing tumor contribute to heterogeneous therapeutic responses. By integrating in vitro and in silico data, we propose a biological scenario where CD19-4-1BB-CAR-T-cells fail to eliminate low-antigen tumor cells, fostering CAR-resistance. These findings offer mechanistic insight into the observed clinical differences between axi-cel (CD28-based) and tisa-cel (4-1BB-based)-treated B-cell lymphoma patients and advance our understanding on CAR-T resistance. Furthermore, we underscore the need for specific FMC63 epitope detection to deliver information on antigen levels accessible for CD19-CAR-T-cells. Visual abstract
    Date: 2025-03-05
    Authors: Krawczyk M, Fernandez-Fuentes N, Fidyt K, Winiarski T, Pepek M, Graczyk-Jarzynka A, Davis J, Bousquets-Muñoz P, Puente XS, Menendez P, Benard E, Wälchli S, Thomas-Tikhonenko A, Winiarska M.
    Ref: bioRxiv
  13. ABSTRACT Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genomes lack a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5′-UTR to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans -acting factors (ITAFs). The cellular protein ITAF 45 (also known as PA2G4 and EBP1) was identified as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for the closely related viruses harboring similar IRES elements such as encephalomyocarditis virus (EMCV) and Theiler’s murine encephalomyelitis virus (TMEV). Here, we demonstrate that ITAF 45 is a pervasive host factor within cells for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF 45 in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF 45 enhances initiation of translation on type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF 45 known to enable binding to viral RNA. These findings challenge previous reports of a unique role for ITAF 45 in FMDV infection, positioning ITAF 45 as a promising antiviral target for various animal viruses and emerging human cardioviruses.
    Date: 2025-03-05
    Authors: Bellucci MA, Amiri M, Berryman S, Moshari A, Owino CO, Luteijn RD, Tuthill TJ, Svitkin Y, Belsham GJ, van Kuppeveld FJ, Sonenberg N.
    Ref: bioRxiv
  14. We present an open source, 3D-printed toolbox for avian embryology. The toolbox includes an electroporation chamber for transfecting functional molecular reagents into developing embryos, and a set of live-imaging chambers, which support avian embryo development while presenting them to a wide variety of microscope setups. We demonstrate both electroporation and imaging chambers by transfecting novel fluorescent reporter constructs for the TGF-beta signalling pathway and Pax7, Brachyury, Cdx2 and Sox2:Oct4 transcription factors into chick embryos and performing time-lapse imaging from stages HH3 - HH12 via both widefield and confocal fluorescence microscopy. Open-source code and ready-to-print STL files are freely available from a GitHub repository in line with FAIR (findability, accessibility, interoperability, and reusability) principles.
    Date: 2025-03-04
    Authors: Antoniou-Kourounioti M, Ramírez de Acuña F, Schettler RCC, Udar SK, Hamzic Petite A, Sivasubramanian RV, Münsterberg AE, Grocott T.
    Ref: bioRxiv
  15. Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) a component of the Wave Regulatory Complex (WRC), one of the most important players in regulating cellular actin dynamics. Interestingly, CYFIP2 transcript undergoes RNA editing, an epitranscriptomic modification catalysed by ADAR enzymes, that leads adenosine (A) to inosine (I) deamination. CYFIP2 editing in the coding sequence results in a K/E substitution at amino acid 320. The functional meaning of this regulation is still unknown. In this study, we aim at investigating the potential implication of CYFIP2 RNA editing related to actin dynamics during cell differentiation, axon development and synaptogenesis in neural cells. We have generated SH-SY5Y neuroblastoma cell lines in which CYFIP2 gene has been functionally inactivated by CRISPR-Cas9 technology. CYFIP2 KO cells showed profound actin filaments disorganisation and loss of the capability to differentiate into a neuronal-like phenotype. Overexpression of both CYFIP2 unedited (K) and edited (E) isoforms rescued normal capability. Finally, we took advantage of primary neuronal culture where endogenous CYFIP2 was knocked down by shRNA technology and CYFIP2 editing variants were overexpressed. While CYFIP2 KD cells reported a decrease in axon development and spine frequency, CYFIP2-E variants increase the number of axon branches, total axon length and dendritic spine frequency compared to either CYFIP2 KD cells or CYFIP-K variants. Overall, our work reveals for the first time a functional significance of the CYFIP2 K/E RNA editing process in regulating the spreading of neuronal axons during the initial stages of in-vitro development and the process of spinogenesis.
    Date: 2025-03-04
    Authors: La Via L, Ndoj E, Bertoli M, Mutti V, Carini G, Filippini A, Bono F, Fiorentini C, Ribaudo G, Gianoncelli A, Borsani G, Russo I, Barbon A.
    Ref: bioRxiv
  16. Abstract The CRISPR-associated endonuclease Streptococcus pyogenes Cas9 (SpCas9) enables site-specific DNA cleavage by transitioning from a pre-catalytic conformation to a catalytically active state, yet how its HNH catalytic domain undergoes an approximately 40 Å displacement towards the target DNA has remained elusive. Here, we combined extensive unbiased molecular dynamics simulations, spanning a cumulative timescale of 160 µs, with Markov state modeling to map the kinetic pathway of SpCas9 activation. In vitro DNA cleavage assays and a cellular fluorescence reporter system further validated the atomic-level mechanisms revealed by our simulations. We found that the folding of the L1 linker and unfolding of the L2 linker serve as the principal driving force, inducing a “gear-and-wedge” cooperative motion within the HNH domain. Concurrently, the REC2 domain moved outward to accommodate the displaced HNH domain and formed transient stabilizing interactions with the HNH domain along the activation route. Site-directed mutagenesis of key L2 linker residues and REC2 loops markedly reduced SpCas9 cleavage efficiency in both HEK293T cells and biochemical assays, underscoring their critical role in SpCas9 ribonucleoprotein activation. Collectively, this study provides a high-resolution view of SpCas9 catalytic activation and opens up new avenues for the rational design of SpCas9 variants with enhanced performance and specificity.
    Date: 2025-03-04
    Authors: Lu S, Li X, Wei J, Chen F, Li M, Liu N, Zhang J.
    Ref: Research Square
  17. The severe acquired respiratory coronavirus–2 (SARS–CoV-2) infection has initiated both acute and chronic COVID–19 disease between 2020 and 2023, currently evolving with other homologous prior coronavirus strains of the Nidoviridae order, which encompasses other prevalent alpha/ beta coronaviruses, but also the Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-1, with recent SARS–CoV–2 variants, increasing demands for effective immunogens and therapeutic approaches that will reduce global disease burden and further infection from SARS–CoV-2 affected individuals that may experience post acute sequelae (PASC) or “Long COVID”. Following a worldwide programme of prophylactic vaccination, there is still a dilemma in the efforts to find prophylactic and early therapeutic approaches that would treat novel SARS-CoV-2 variants and prevent future epidemics or pandemics within host human and animal populations, where zoonotic or cross species transfer naturally occurs. Concerns about viral immune escape intersect at a specific point; a gained evolutionary ability of several viruses to co–infect and compete against previous scientific advances since 1796 that remain undetected or asymptomatic during the early stages of infection progressing to symptomatic and severe disease via the double methylation of the 5' end of eukaryotic DNA or RNA-based viral genomes, the 7-MeGpppA2’-O-Me cap, and its double methylation capping process is performed by the activated viral 2’ - O - Methyltransferase (MTase) enzyme, a complex of two viral non-structural proteins (NSPs) joined together through an activation process (NSP10/16) and by N7-Methyltransferase (N7-MTase/NSP14), respectively. Moreover, it was discovered that polymorphic viruses translate NSP1, which prevents the activation of various Pattern Recognition Receptors (PRRs), and consequently, detection of Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs) alike. NSP1 also silences important interferon-encoding genes (INGs) and interferon-stimulated genes (ISGs), is signalled in a paracrine manner to neighbouring cells, and that induces the apoptosis of host cells, inducing an effect of “trace erase” effect and making the viral infection as immunologically “invisible” as possible during the initial, key stages of viral replication and distribution, all such mechanisms occurring independently of the viruses in cause. Another important viral NSP is NSP14, as it plays two functional roles that are independent of each other; to produce new viral genetic material for the purpose of maintaining the validity of the viral genome as well, and not just transfer a methyl group to the 5’ end of the viral genome. Other viral NSPs share a role with NSP1, 10, 14 and 16 in directly suppressing the activation of PRRs and ISGs, and all such viral proteins help the virus in its process of self-camouflaging against first- and second-line immunity, thereby often severely impacting the quality of the produced adaptive immune responses. The outcome of all such phenomena is the sharp decrease in the host Type I and Type III interferons' (IFNs) rate of synthesis by the host cells, that would usually occur and affect homeostatic cellular pathways, resulting in further viral replication and induced apoptosis. Nonetheless, effects of microbial immune evasion during the development of other viral or carcinogenic pathologies are not widely known. In short, polymorphic viruses developed a proportionate evolutionary response against developed adaptive immune responses, by currently relying on gaps mostly situated in the natural immune system in their process of molecular self-camouflaging. Scientists developed numerous approaches of early treatment that generally showed good success rates and fewer risks of adverse events, and the still early present stages of COVID-19 research should also be taken into consideration whilst filtering for the most appropriate solutions. For example, the administration of recombinant human interferons I and III into the nasal mucosa cellular layer, as key mediators of anti–viral activity, can simulate intracellular infection and stimulate cellular activity in a timely manner, training the innate and adaptive immune system cells to develop and appropriately stimulate an adequate immune response through B and T cells. Another example could involve the treatment of natural and adaptive lymphocytes with a low dose of IFNs I and possibly III, prior to their insertion into the host lymphatic system, possibly alongside additional recruitment of plasmacytoid dendritic cells (pDCs) as further interferon “factories”, all with the purpose of early infection management. It might be that focusing on directly offering the immune system the information about the genetics and protein structure of the pathogen, rather than training its first-line mechanisms to develop faster, excessively increases its specificity, making it reach a level that brings the virus the opportunity to evolve and escape previously-developed host immune mechanisms. It is until the scientific community realises this potentially crucial aspect that large proportions of the world population will probably continue to face serious epidemics and pandemics of respiratory diseases over the coming several decades, evidenced with dengue fever and more recently, monkeypox and possibly avian flu. Of note, it has been indicated that IFN I and / or III display significant immunising, early therapeutic and clinical disease onset-attenuating effects for many other microbial evoked diseases, as well as for a number of oncological diseases. Microbial agents could undergo loss-of-function research upon genes responsible for inducing clinical illness whilst keeping genes responsible for microbial reproduction and transmission at least generally as functional, CRISPR-Cas9 genome editing to have genes encoding proteins suppressive of the host interferon system eliminated prior to human genes encoding Pattern Recognition Receptor activator or agonist proteins, such as outer membrane proteins of Neisseria meningitidis, as well as Type I, Type III and possibly even Type IV Interferons and various ISGs inserted into the microbial genome. Such an approach would be based upon the model of editing genes of harmless bacteria to transform such them into “producers” and “distributors” of human insulin, and could turn several microbial agents into clinically harmless, transmissible “factories” for various key elements of the host interferon system, potentially placing such microbes into a reverse evolutionary path that would be deemed as “natural de-selection”, visibly reducing the average burden of disease and metabolic stresses, which in turn could gradually increase average human and animal lifespans worldwide.
    Date: 2025-03-03
    Authors: Carp T, Metoudi M, Ojha V.
    Ref: Preprints.org
  18. ABSTRACT Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae , a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.
    Date: 2025-03-03
    Authors: Smidler AL, Marrogi EA, Scott S, Mameli E, Abernathy D, Akbari OS, Church GM, Catteruccia F, Esvelt K.
    Ref: bioRxiv
  19. Summary Sex chromosomes shape male (XY) - female (XX) differences in development and disease. These differences can be modelled in vitro by comparing XY and XX human induced pluripotent stem cells (hiPSCs). However, in this system, inter-individual autosomal variation and unstable X-dosage compensation can confound identification of sex chromosomal effects. Here, we utilise sex chromosome loss in XXY fibroblasts to generate XX and XY hiPSCs that are autosomally isogenic and exhibit stable X-dosage compensation. We also create X-monosomic (XO) hiPSCs, to investigate X-Y dosage effects. Using these autosomally isogenic lines, we examine sex differences in pluripotent stem cell expression. Transcriptional differences between XX and XY hiPSCs are surprisingly modest. However, X-haploinsufficiency induces transcriptional deregulation predominantly affecting autosomes. This effect is mediated by Y-genes with broad housekeeping functions that have X-homologues escaping X-inactivation. Our isogenic hiPSC lines provide a resource for exploring sex chromosome effects on development and disease in vitro .
    Date: 2025-03-03
    Authors: Meleckyte R, Varsally W, Zohren J, Eriksson J, Incitti T, Starnes L, Pointon A, Hicks R, Powell BE, Turner JM.
    Ref: bioRxiv
  20. ABSTRACT Tuberculosis (TB) and COVID-19 are leading infectious diseases with high mortality, caused by Mycobacterium tuberculosis ( Mtb ) and SARS-CoV-2 (SC2) , respectively. Co-infection is common but is often undiagnosed as it is challenging to process both pathogens from a single sample. In this study, we present a simple and efficient method for co-extracting nucleic acids (NA) from these two distinct respiratory pathogens for downstream diagnostic testing. We evaluated three different nucleic acid amplification (NAA)-based platforms, LightCycler480 (LC480) qPCR, Qiacuity digital PCR (dPCR), and Cytation3 for CRISPR-Cas13a-based SHINE-TB/SC2 detection assays. Chelex-100 chelating resin-based boiling preparation method was optimized for Mtb NA extraction from saliva and sputum. Saliva showed compatibility with all three platforms, with sensitivity as low as 100 CFU/ml (or 2 genomic copies/µl). This method worked well for sputum using dPCR at 100% (21/21) positivity, though the CRISPR-based SHINE-TB assay showed more variability and sensitivity to sputum inhibitor carry-over, resulting in an 81% positive rate (17/21). Diluting sputum with TE buffer (1:1) improved the detection (2/4). Extraction efficiency of our method was 48%, 62.2%, 86.4% and 99.3% for concentrations 10 5 , 10 4 , 10 3 and 10 CFU/ml, respectively. The dynamic range for Mtb spiked in pooled sputum showed 100% detection (N=8) at ≥10 3 CFU/ml with all three methods. Dual-pathogen co-extraction and detection of SC2 (10 5 PFU/ml) and Mtb (10 5 CFU/ml) in salivary sputum was successful using CRISPR-Cas13a assays. We have developed a rapid and efficient co-extraction method for multi-pathogen testing across diagnostic platforms and believe this is the first protocol optimized to co-extract Mtb and SARS-CoV-2 from a single sample.
    Date: 2025-03-03
    Authors: Modi NH, Dunkley ORS, Bell AG, Hennig E, Wats A, Huang Y, Daivaa N, Myhrvold C, Xie YL, Banada P.
    Ref: medRxiv
  21. The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains poses serious challenges to global tuberculosis control, highlighting the urgent need to unravel the mechanisms underlying multidrug resistance. In this study, we screened for spontaneous bortezomib (BTZ)-resistant mutants of Mycobacterium smegmatis and identified a strain, Msm-R1-2, which exhibited high-level resistance to both BTZ and linezolid. Whole-genome sequencing revealed mutations in MSMEG_1380 and MSMEG_0965 genes, encoding a transcriptional regulator (involved in regulating efflux pump expression) and a porin, respectively are potential contributors to drug resistance. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that dual mutations in MSMEG_1380 and MSMEG_0965 synergistically enhanced resistance to BTZ and LZD, conferring cross-resistance to other antibiotics, including moxifloxacin and clofazimine. Ethidium bromide accumulation assay demonstrated that mutations in MSMEG_0965 reduce cell wall permeability, contributing to multidrug resistance. Furthermore, previous studies have shown that mutations in MSMEG_1380 upregulate the mmpS5-mmpL5 efflux system, thereby promoting drug efflux and reducing intracellular drug concentrations, while mutations in MSMEG_0965 impair porin function, limiting antibiotic uptake and significantly contributing to the multidrug-resistant phenotype. Collectively, these findings provide valuable insights into the molecular mechanisms of mycobacterial drug resistance, underscoring the pivotal roles of efflux and uptake pathways in the development of multidrug resistance.
    Date: 2025-03-03
    Authors: Zhang H, Fang C, Yusuf B, Zhu X, Wang S, Hameed HA, Gao Y, Zhang T.
    Ref: Preprints.org
  22. Understanding how cellular pathways interact is crucial for treating complex diseases like cancer, yet our ability to map these connections systematically remains limited. Individual gene-gene interaction studies have provided insights 1,2 , but they miss the emergent properties of pathways working together. To address this challenge, we developed a multi-gene approach to pathway mapping and applied it to CRISPR data from the Cancer Dependency Map 3 . Our analysis of the electron transport chain revealed certain blood cancers, including acute myeloid leukemia (AML), depend on an unexpected link between Complex II and purine metabolism. Through stable isotope metabolomic tracing, we found that Complex II directly supports de novo purine biosynthesis and exogenous purines rescue AML from Complex II inhibition. The mechanism involves a metabolic circuit where glutamine provides nitrogen to build the purine ring, producing glutamate that Complex II must oxidize to sustain purine synthesis. This connection translated to a metabolic vulnerability whereby increasing intracellular glutamate levels suppresses purine production and sensitizes AML to Complex II inhibition. In mouse models, targeting Complex II triggered rapid disease regression and extended survival in aggressive AML. The clinical relevance of this pathway emerged in human studies, where higher Complex II gene expression correlates with both resistance to mitochondria-targeted therapies and worse survival outcomes specifically in AML patients. These findings establish Complex II as a central regulator of de novo purine biosynthesis and identify it as a promising therapeutic target in AML.
    Date: 2025-03-02
    Authors: Stewart AE, Zachman DK, Castellano-Escuder P, Kelly LM, Zolyomi B, Aiduk MD, Delaney CD, Lock IC, Bosc C, Bradley J, Killarney ST, Ilkayeva OR, Newgard CB, Chandel NS, Puissant A, Wood KC, Hirschey MD.
    Ref: bioRxiv
  23. CRISPR/Cas9 is a powerful tool for targeted genome engineering experiments. With CRISPR/Cas9, genes can be deleted or modified by inserting small peptides, fluorescent proteins or other tags for protein labelling experiments. Such experiments are important for detailed protein characterization in vivo . However, designing and cloning the corresponding constructs can be repetitive, time consuming and laborious. To aid users in CRISPR/Cas9-based genome engineering experiments, we built CrisprBuildr, a web-based application that allows users to delete genes or insert fluorescent proteins at the N- or C-terminus of their gene of choice. The application is built on the Drosophila melanogaster genome but can be used as a template for other available genomes. We have also generated new tagging vectors, using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments. CrisprBuildr guides users through the process of designing guide RNAs and repair template vectors. CrisprBuildr is an open-source application and future releases could incorporate additional tagging or deletion vectors, genomes or CRISPR applications.
    Date: 2025-03-02
    Authors: Horsley N, Barnau Sythoff Av, Delgado M, Liu S, Cabernard C.
    Ref: bioRxiv
  24. Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo . These data reveal novel regulators that link plasma cell secretory capacity and lifespan. Summary Long-lived plasma cells rely on V-type ATPases, PI4K, DDX3X, and MYD88 signals for maximal secretory capacity and survival
    Date: 2025-03-02
    Authors: D’Souza LJ, Young JN, Coffman H, Petrow EP, Bhattacharya D.
    Ref: bioRxiv
  25. Background Colon cancer progression heavily relies on intricate mechanisms of invasion, metastasis, and migration. Tight junction protein Cldn2 has emerged as a potential regulator of these processes. This study aimed to elucidate the molecular mechanisms linking Clan2 deletion to gene expression changes related to motility, invasion, and metastasis in colon caner. Methods CRISPR/Cas9-mediated knockout of human Cldn2 in HCT116 cells was conducted, and the resulting cells were compared to the wild-type cells using real-time PCR to analyze the expression of genes associated with invasion and metastasis. Results Cldn2-KO resulted in a widespread downregulation of genes linked to motility, invasion, and metastasis, including ZONAB, NDRG1, Cldn14, Cldn23, Bcl2, , P53, and BCL-6. These findings suggest a potential regulatory role of Cldn2 in the expression of these genes, influencing colon cancer cell migration and spread. Conclusion This study identified Claudin-2 as a crucial regulator of genes involved in colorectal cancer metastasis. Downregulation of these genes upon Claudin-2 deletion suggests its inhibitory role in cancer cell motility and invasion. Further investigation into the specific downstream signaling pathways mediated by Claudin-2 could pave the way for novel therapeutic strategies targeting metastasis inhibition.
    Date: 2025-03-01
    Authors: Alghamdi RA, Al-Zahrani MH.
    Ref: bioRxiv
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