Artificial intelligence predicts genetic network disruptions to overcome CAR-T resistance in Mantle Cell Lymphoma

Loss-of-function alterations in TRAF3 drive BTK/PLCG2-independent ibrutinib resistance in CLL

CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.

Molecular diversity and functional roles of fibrillarin: Comparative insights across trypanosomatids and other model organisms.

Cyclin-Dependent Kinase 9 (CDK9) is a critical regulator of transcriptional elongation, functioning within the Positive Transcription Elongation Factor b (P-TEFb) complex alongside Cyclin T1. P-TEFb facilitates the release of RNA polymerase II (RNAPII) from promoter-proximal pausing, thereby enabling productive transcriptional elongation. CDK9 activity is tightly controlled by the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, comprising 7SK snRNA, LARP7, MEPCE, and HEXIM1/2. Under homeostatic conditions, the 7SK snRNP sequesters and inactivates a fraction of P-TEFb, maintaining it in a repressed state. However, in response to cellular stress or increased transcriptional demand, P-TEFb is released from 7SK snRNP, activating CDK9 to ensure precise, context-dependent transcriptional control. This regulatory switch allows dynamic adaptation to environmental and intracellular cues. Emerging evidence implicates 7SK snRNP deregulation in cancer progression. This review explores the intricate interplay between 7SK snRNP and CDK9, highlighting how disruptions in individual 7SK snRNP components drive transcriptional imbalances, amplify oncogenic programs, and promote a tumorigenic environment.

Single-step purification of functional Cas9 protein via the ubiquitin expression system.

Abstract Description The functional specialization of CD4 T lymphocytes into various subtypes, including TH1 and TFH cells, is crucial for effective immune responses. TFH cells facilitate B cell differentiation within germinal centers, while TH1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of integrin α4 in regulating TFH and TH1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating DNA fragments. By analyzing the effects of Itga4 deficiency on TFH and TH1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the TH1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting. Funding Sources Supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) No. 2021R1F1A1060347, 2023R1A2C1007319, and RS-2023-00258956, NIH P01AI145815 (sub-award) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Inhibition of acid sphingomyelinase increases SMN levels and connects sphingolipid metabolism to Spinal Muscular Atrophy.

In this study, we established a ribonucleoprotein-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome co-editing method for the white koji fungus, Aspergillus luchuensis mut. kawachii. To introduce the single guide RNA-Cas9 ribonucleoprotein complex into protoplast cells of A. luchuensis mut. kawachii, we investigated the conditions for protoplast preparation using Yatalase -Plus-. Subsequently, we employed the ribonucleoprotein-based method to knockout the ATP sulfurylase-encoding sC gene, which imparts selenate resistance in the model strain NBRC 4308 and the industrial strain No. 8046. Furthermore, we explored genome co-editing by simultaneously targeting sC along with either the orotidine 5'-phosphate decarboxylase-encoding pyrG gene or the transcriptional activator of protease genes-encoding prtR gene in NBRC 4308. The transformants were selected in medium containing selenate, resulting in the successful generation of pyrG- and prtR-knockout strains. Similarly, transformants were selected on medium containing selenate, resulting in the successful generation of prtR-knockout strain in No. 8046. These results demonstrate that the ribonucleoprotein-based genome co-editing method is applicable not only to the model strain but also to industrial strains, making it a promising approach for manipulating A. luchuensis mut. kawachii.

Abstract Description Several studies have shown that nucleofection can be used to edit human myeloid cells. However, nucleofection of myeloid cells has many disadvantages, including low viability and editing efficiency. The aim of this study is to develop a robust system to genetically engineer primary human monocyte-derived macrophages and dendritic cells (DCs), while maintaining their functional integrity. We tested several in vitro approaches to deliver ribonucleoproteins (RNPs). Using Beta-2 microglobulin (β2M) as a model gene target, nucleofection protocols to deliver RNPs yielded high editing efficiency (90-100%) at the genomic, transcriptional and protein levels. However, nucleofection severely compromised the functional integrity of the cells as they failed to secrete interleukin-1 beta in response to NLRP3 inflammasome activation. We tested alternative RNP delivery methods, including cell-penetrating peptides and lipid nanoparticles. However, the editing efficiencies only reached approximately 30% and resulted in a significant loss in viability. As a solution, we adapted protocols from the Doudna lab to use Enveloped Delivery Vehicles (EDVs) to deliver RNP complexes for gene editing. We reached up to 90% editing efficiency in both primary human macrophages and DCs. We are in the process of validating the functional integrity of EDV-edited myeloid cells and the transcriptional impact of EDV delivery. In conclusion, EDVs are robust and programmable gene editing tools for myeloid cells. Funding Sources NIH R01AI175614 Topic Categories Technological Innovations in Immunology (TECH)

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric “intasome” assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the IN tetramer is an important antiviral target for investigational allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions and suggest a working model for IN-viral RNA binding. Moreover, our structure of the IN tetramer provides atomic blueprints for the rational development of improved allosteric inhibitors.

BackgroundTelomeres are known to be important for Leishmania biology but the mechanistic of how the process of telomere maintenance contributes to genome stability remains an unanswered question. Their maintenance is most commonly facilitated by the telomerase ribonucleoprotein complex that elongates telomeres countering their natural shortening due to the incomplete DNA replication in each cell cycle. In some organisms, telomere maintenance is achieved through telomerase-independent mechanisms, such as the Alternative Lengthening of Telomeres (ALT) pathways described in yeasts with dysfunctional telomerase and some rare telomerase-negative human cancer cells. Molecular markers for the ALT pathway include presence of the heterogeneous (in their length and sequence) telomeres, high level of telomeric exchange between the sister chromatids, increased expression of Rad51 and associated proteins, and occurrence of extrachromosomal telomeric repeats that can be present in either linear or circular form.ResultsHere, we used third-generation sequencing techniques in combination with other approaches and analyzed telomeres of L. mexicana at unprecedented high-level resolution. We demonstrate that Ku80 ablation-driven telomere elongation varies between chromosomes, possibly due to the chromosome-specific recombination rates, which are sequence/content dependent and associated with the structure of the telomeric tandemly repeated sequence, TTAGGG. Moreover, this telomere length heterogeneity is accompanied by an increased level of C-circles, a subclass of circular telomeric DNA highly specific for ALT activity.ConclusionsOur findings underscore that L. mexicana promastigotes have an inherent ability to utilize ALT, and the loss of Ku80 and/or TERT further enhanced this trait. These proteins work together to maintain telomere integrity, inhibit recombination, and stabilize telomere lengths. Our data suggest that ALT may be a fundamental and readily activated feature of Leishmania biology, and that telomere regulation in this organism significantly differs from what has been observed in other eukaryotic model species, including iconic T. brucei.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12154-z.

Influenza A virus (IAV) forms pleomorphic particles that package eight ribonucleoprotein complexes (vRNPs), each carrying a distinct RNA genome segment. vRNPs assemble in the nucleus and undergo selective sorting during Rab11a-mediated trafficking to the plasma membrane. Virion assembly is orchestrated by matrix protein 1 (M1), which forms a layer beneath the viral envelope containing hemagglutinin (HA) and neuraminidase (NA). However, molecular details of vRNP distribution, cytosolic trafficking, and coordination of IAV assembly remains unclear. Using in situ cryo-ET, we reveal that HA-containing membranes provide Rab11a-dependent platforms for membrane-assisted vRNP clustering, reducing inter-vRNP distances. In the absence of HA, vRNPs cluster on NA-containing membranes and virus assembly remains intact, indicating that vRNP clustering and trafficking is membrane-assisted but HA independent. The characteristic 7 + 1 vRNP bundle forms concomitantly with budding and is orchestrated by M1 layer assembly that precedes plasma membrane attachment. We further reveal that intracellular M1 forms multilayered helical assemblies of antiparallel dimers, structurally distinct from the M1 layer in virions. These assemblies are compact in the nucleus but partially dissociate in the cytoplasm, likely serving as a reservoir for budding. Together, our findings uncover membrane-assisted vRNP clustering and molecular details of M1 coordinated influenza virus assembly.

Telomerase is a ribonucleoprotein complex that elongates telomeric DNA, ensuring germline immortality. In this study, we identified the Caenorhabditis elegans telomerase RNA component 1 (terc-1), as the first known telomerase RNA expressed as an intronic long noncoding RNA (lncRNA), embedded in an intron of germline-up-regulated gene nmy-2. terc-1 undergoes splicing, polyadenylation, and nuclear RNA exosome-dependent maturation, stabilized by H/ACA small nucleolar ribonucleoproteins, thus co-opting the H/ACA small nucleolar RNA (snoRNA) biogenesis machinery. Mutations in terc-1 led to progressive telomere shortening and sterility in successive generations. Artificially transplanting the nmy-2 intron into the introns of germline-expressed genes but not non-germline-expressed genes restored germline immortality, highlighting the importance of genomic context. Our findings suggest that nematode telomerase RNA is a snoRNA-like intronic lncRNA that exploits the introns of germline-up-regulated genes to ensure species survival.

Double-stranded RNA (dsRNA), which induces an innate immune response against viral infections, is rarely detected in influenza A virus (IAV)-infected cells. Nevertheless, we previously reported that the influenza A viral ribonucleoprotein (vRNP) complex generates looped dsRNAs during RNA synthesis in vitro. This finding suggests that IAV possesses a specific mechanism for sequestering dsRNA within infected cells, thereby enabling viral evasion of the innate immune response. Here, we found that dsRNAs were detected in infected cells lacking the expression of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both encoded by the same RNA segment. Indeed, the looped dsRNA-vRNP complexes were isolated from IAV-infected cells. Interestingly, NS1 molecules masked the entire looped dsRNA generated by vRNP in vitro, implying a potential role for NS1 in segregating viral dsRNA from cytoplasmic dsRNA sensors. Furthermore, dsRNAs were sequestered within the nucleus of wild-type IAV-infected cells, whereas their translocation to the cytoplasm was observed in NS1-deleted mutant virus-infected cells expressing M1 and NEP. This result indicates the possibility that dsRNA is transported to the cytoplasm in association with vRNP. Notably, the cytoplasmic translocation of dsRNA triggered the nuclear translocation of interferon regulatory factor 3, suggesting the capability of dsRNA in inducing the innate immune response. These findings highlight IAV's distinctive strategy for circumventing innate immunity by sequestration of dsRNAs.IMPORTANCEIt is widely recognized that double-stranded RNA (dsRNA) produced during viral infection triggers an innate immune response. However, the influenza A virus (IAV) has been thought to rarely produce dsRNA within infected cells. Here, we detected dsRNA in the nucleus of IAV-infected cells which lacked the expression of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both encoded by a single RNA segment. High-speed atomic force microscopy demonstrated that NS1 entirely concealed dsRNA produced by the viral ribonucleoprotein complexes, thereby segregating it from cytoplasmic dsRNA sensors that trigger the innate immune response. Interestingly, cytoplasmic translocation of dsRNA was observed in cells infected with an NS1-deleted mutant virus, where M1 and NEP were expressed, resulting in the nuclear translocation of interferon regulatory factor 3. Collectively, our findings suggest that IAV adeptly sequesters dsRNA to evade the innate immune system.

Influenza A virus (IAV) poses a major threat to global public health, exerting immense pressure on human health and the economy. The IAV nucleoprotein (NP) is an ideal target for antiviral drug development. Through Mini-genome and Surface Plasmon Resonance assays, this study discovered and verified that mycophenolic acid methyl ester (MAE), a secondary metabolite produced by the marine algal-associated fungus Phaeosphaeria spartinae, can target the viral nucleoprotein to exert anti-IAV activity. Pull-down assays and immunofluorescence have revealed that MAE blocks the nuclear import of viral ribonucleoprotein complexes (vRNP) by interfering with the interaction between NP and IMP-α. It also affects the vRNP assembly process by regulating NP oligomerization and the interaction between NP and PB2. In addition, Sandwich ELISA and Electron Microscopy experiments showed that MAE can also inactivate viral particles to reduce the risk of infection. Comprehensive research results indicate that MAE exerts its effects by inhibiting the viral NP protein, which has laid an important foundation for the development of marine-derived NP-targeted drugs.

Microalgae biotechnology has witnessed a significant transformation with the advent of CRISPR/Cas genome editing technologies, which enabled precise gene alterations that were unattainable using conventional methods. Traditional transformation techniques have advanced to CRISPR-based platforms, mainly due to significant developments in delivery methods like as systems based on nanoparticles and RNP (ribonucleoprotein complexes), which overcame species-dependent barriers and demonstrated the innovative influence of CRISPR technology on genetic engineering of microalgal systems over traditional methods. Effective metabolic engineering advancements for improved pigment, lipid and biomolecule synthesis are crucial approaches. For this, comprehensive analyses of gene deletions or knockouts, overexpression and regulatory changes from model organisms like Chlamydomonas reinhardtii to economically important organisms like Nannochloropsis are important. This study highlights elevated expression of biosynthetic genes and suppression of competitive pathways, along with significant developments in multiplexed genome editing, which enable coordinated pathway alterations by precisely targeting many genes. Despite novel approaches, existing constraints like as off-target consequences, regulatory barriers and editing efficiency constraints are critically evaluated for enhancement of CRISPR technology in microalgal modification. Revolutionizing developments such as optogenetics-CRISPR inclusion, pan-genomic genetic modifications of microalgal populations and AI-driven autonomous genome engineering will establish engineering microalgae as leading platforms for sustainable biotechnology.

Disclosure: J. Banik: None. J. Lim: None. C. Bolen: None. M. Reddick: None. S. Byrum: None. L. Hardy: None. Y. Rahmatallah: None. A.K. Odle: None. G.V. Childs: None. A.M. MacNicol: None. M.C. MacNicol: None.The anterior pituitary regulates multiple organismal functions including growth and development, reproduction and lactation, and metabolism and stress response. Distinct cell populations within the pituitary are required to synthesize and secrete the different hormones that direct these disparate regulatory processes. An insufficiency, or excess, of cells in any population contributes to multiple pathophysiological conditions. The mechanisms by which the distinct cell populations lineages are maintained is not fully understood. Here, we tested a requirement for the stem cell and progenitor cell marker, Musashi, in maintenance of pituitary cell populations. This study utilized a mouse genetic model in which the Musashi1 gene (Msi1) and the Musashi2 gene (Msi2) are deleted in all pituitary hormone producing cell type through use of a Prop1 promoter-driven Cre-lox system. Initial proteogenomic analysis of pituitary from adult male Msi1Msi2-null mice versus control littermates, utilized both mass spectrometry and RNA sequencing from the same sample. Differential expression analyses show a broader impact on the transcriptome than on the proteome profile in Musashi-null mutants. Correlation analysis of differential mRNA expression and differential protein abundance indicates that Musashi represses translation of transcripts that are involved in mRNA processing and localization, and ribonucleoprotein complex assembly and biogenesis, and hormone secretion and transport, all of which would be required for maintenance of hormone-producing pituitary cell populations. Notably, the transcriptomic findings indicate significant increases in a subset of pituitary cell populations e.g. somatotropes (Gh), lactotropes (Prl) and thyrotropes (Tshb), along with a significant decrease in other pituitary cell populations e.g. gonadotropes (Gnrhr), progenitor cells (Pou1f1), and stem cells (Sox2). These findings support a model in which Musashi mediates maintenance of a stem/progenitor pool to ensure an appropriate balance of pituitary cell populations.Presentation: Saturday, July 12, 2025

Creation of humanized genetically modified animals for biomedical research is an important direction in the activity of the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency (FMBA) of Russia. Since 2013, the Center has obtained more than 20 lines of transgenic, knockout, and transgenically-knockout mouse biomodels using classical transgenesis and the plasmid version of the CRISPR/Cas9 technology. The main specialized lines involve humanized transgenic mouse biomodels carrying alleles HLA class I — HLA-В*, HLA-В**, HLA-В***, HLA-С*, HLA-B*07:02, HLA-С*07:02, HLA-A*02:01, HLA-В*** КО, HLA-A** КО, HLA-B**** КО, HLA-С** КО, as well as NAT1, NAT2, АСЕ2 hom, hACE2-Tom (HDRKI), SMN2 (S2), PrPSc, АСЕ2 hom / HLA-C**, β2m mus KO, PrP КО, STE24 КО, SMN (S6) КО, SMN2 КО, PrP КО / PrPSc, NAT1 KO, NAT2 KO. In this article, we review the genetic constructs and methods used to produce genetically modified animals. The article summarizes the long-term experience of using the plasmid variant and discusses the need to switch to the CRISPR/Cas9 ribonucleoprotein complex in the view of its high efficiency and specificity, high frequency of targeted modifications, the ability to use multiple gRNAs, and the insertion of a single copy of the HDR-DNA template (transgene). We also explore the possibility of replacing imported reagents and kits with domestic products.

Age-related macular degeneration (AMD), a leading cause of vision loss in elderly individuals, is a multifactorial disease driven by genetic, environmental, and cellular aging processes. Emerging evidence highlights the critical role of ribonucleic acid (RNA) splicing dysfunction in AMD pathogenesis, with a focus on the U1 small nuclear ribonucleoprotein (U1 snRNP) complex, a key spliceosome component. U1 snRNPs ensure the fidelity of RNA cotranscription and pre-mRNA splicing initiation, and their dysfunction has been implicated in neurodegenerative disorders and other age-related diseases. This narrative review explores the impact of U1 snRNP dysregulation on retinal cells, focusing on its role in transcriptomic instability, impaired protein homeostasis, cellular stress, impaired autophagy, and inflammation, which are important features of AMD pathogenesis. Finally, we propose that targeting U1 snRNP dysfunction could provide a novel therapeutic approach to slow, prevent, or restore retinal degeneration, offering insights into broader implications for age-related diseases. Understanding the molecular mechanisms underlying U1 snRNP dynamics in retinal health and degeneration is essential for developing innovative and effective treatments for AMD, which may provide ways to delay or reverse the effects of aging and associated diseases.