Profiling antigens presented on MHC class I molecules on the cell surface is essential to identify candidate antigens for targeted and personalized immunotherapies. However, mass spectrometry-based immunopeptidomics has traditionally been limited by high input requirements, extensive sample manipulation, and expensive reagents. To overcome these challenges, we developed MHC1-TIP: a scalable, single-tube and cost-effective workflow to enable robust MHC-I ligandome recovery from cell lines, patient-derived organoids, and sub-milligram amounts of clinical tissues. Moreover, MHC1-TIP also preserves compatibility with additional omics profiling technologies and we demonstrate its capacity for quantitative and multimodal profiling of the proteome and immunopeptidome from the same sample to enable integrated analyses of protein expression and antigen presentation. Application of MHC1-TIP to primary renal cell carcinoma fragments revealed extensive intratumoral heterogeneity in antigen presentation that was poorly correlated with source protein expression. MHC1-TIP represents a broadly applicable and sensitive approach for low-input, multimodal immunopeptidomics with clinical and translational relevance.

Maintaining intestinal homeostasis relies on the intricate interplay among the mucosal epithelium, immune system, and host microbiome. A key question is how innate immune cells sense and process microbes in the gut lumen, eliciting appropriate protective responses without causing tissue injury. Clearance of invading microbes and initiation of downstream inflammatory responses are central to this process and require proper function of the endolysosomal system. Dysfunction of this system can predispose the host to chronic inflammatory disorders and acute infections. Here, through forward genetic screening of N-ethyl-N-nitrosourea (ENU)-mutagenized mice and CRISPR/Cas9 validation, we identify Lrmda , encoding leucine-rich melanocyte differentiation-associated protein (LRMDA), as a key regulator of intestinal homeostasis. Using hematopoietic chimera and conditional knockouts, we show that LRMDA functions primarily in CD11c + cells, including mucosal dendritic cells (DCs) and macrophages, but not in non-hematopoietic cells. Proteomic, cellular, and biochemical analyses reveal that LRMDA directly and cooperatively interacts with the endolysosome-specific small GTPase Rab32 and the endosomal recycling complex Retriever. Loss of LRMDA or Retriever function increases susceptibility to dextran sodium sulfate (DSS)-induced colitis and impairs clearance of Listeria monocytogenes . Together, our findings establish the Rab32-LRMDA-Retriever complex as a critical regulator of endolysosomal trafficking in innate immune cells, essential for maintaining intestinal immune homeostasis.

ABSTRACT BACKGROUND While mitochondrial dysfunction clearly drives cardiac deterioration in major heart diseases, the mechanisms controlling mitochondrial quality remain incompletely understood. This study investigated whether TIGAR (TP53-induced glycolysis and apoptosis regulator) deficiency influences cardiac protection through mitochondrial quality control pathways. METHODS We generated both whole-body and cardiomyocyte-specific TIGAR knockout mice that were assessed for cardiac function following myocardial infarction (induced by left anterior descending coronary artery ligation) and diet-induced cardiomyopathy (using a 6-month high-fat diet protocol). Mitochondrial quality control was evaluated through mitophagy assays, subcellular fractionation, and molecular analyses. Epigenetic regulation was assessed using whole-genome bisulfite sequencing, chromatin immunoprecipitation, and CRISPR-mediated gene editing in multiple cell lines. RESULTS Both whole-body (TKO) and cardiomyocyte-specific (hTKO) TIGAR knockout mice demonstrated cardioprotection following myocardial infarction. These animals maintained significantly better ejection fraction (43.35±17.76% vs 26.36±9.83% in wild-type controls, P<0.05) and displayed complete resistance to diet-induced cardiac hypertrophy, despite comparable weight gain. TIGAR deficiency led to dramatic increases in Parkin expression across multiple tissues, 6-fold increases in heart and muscle, and 5-fold increases in brain, which enhanced mitophagic responses during metabolic stress conditions including fasting and high-fat diet feeding. Generation of Parkin/TIGAR double knockout mice eliminated this protection, confirming Parkin’s essential role. Notably, adult manipulation of TIGAR through viral overexpression or knockdown failed to alter Parkin levels, suggesting developmental programming. Whole-genome bisulfite sequencing revealed reduced DNA methylation in a specific 3.2 kb region within Parkin gene intron 10, and CRISPR deletion of this regulatory region increased Parkin expression 10-fold in C2C12 myoblasts and 6-fold in 3T3-L1 fibroblasts. CONCLUSIONS These findings reveal a novel TIGAR-Parkin regulatory axis operating through epigenetic mechanisms during cardiac development to establish lifelong cardioprotection via enhanced mitochondrial quality control. This discovery points toward new therapeutic approaches targeting developmental metabolic programming for heart disease prevention and identifies specific epigenetic targets for cardiovascular therapy. CLINICAL PERSPECTIVE What Is New? TIGAR deficiency establishes lifelong cardiac protection through developmental epigenetic programming of Parkin expression. A novel 3.2 kb differentially methylated region in Parkin intron 10 regulates mitochondrial quality control in the heart. Early metabolic programming during cardiac development can establish permanent cardioprotective phenotypes. The TIGAR-Parkin axis provides protection against both acute ischemic injury and chronic metabolic cardiomyopathy. What Are the Clinical Implications? Targeting the TIGAR-Parkin pathway could provide novel therapeutic approaches for preventing both ischemic heart disease and diabetic cardiomyopathy. Early developmental interventions targeting cardiac metabolism might establish lifelong cardiovascular protection. Epigenetic modifications of mitochondrial quality control genes represent potential therapeutic targets. The findings suggest optimal timing for cardiovascular preventive interventions may be during critical developmental windows.

The entomopathogenic nematodes (EPN) of the genus Steinernema serves as a valuable experimental model for studying microbial symbiosis and is economically important as organic pest control agent in agriculture. Although most Steinernema species are dioecious (male-female), consistent genetic manipulation has thus far only been demonstrated in the hermaphroditic species Steinernema hermaphroditum . In this study, we adapted a CRISPR-Cas9–based gene editing approach to Steinernema feltiae , a dioecious species widely used in agricultural applications. Using gonadal microinjection, we targeted the conserved gene unc-22 in S. feltiae and generated stable mutant lines with large on-target deletions. Mating tests revealed that Sf-unc-22 is X-linked and exhibits a conditionally dominant twitching phenotype. Additionally, Sf-unc-22 mutants display distinct body morphology compared to wild-type nematodes. Homozygous mutant lines can be reliably maintained through cryopreservation. Altogether, our work provides a proof of concept that genetic tools developed in S. hermaphroditum can be effectively adapted to other agriculturally relevant and dioecious Steinernema species— broadening the scope of molecular genetic research in microbial ecology and enhancing their potential applications in agriculture.

During development, neurons are produced in excess and those that receive trophic support are maintained, whereas excess neurons are eliminated, enabling the establishment of appropriate neural circuits. In vertebrates, neurotrophin ligands promote cell survival during periods of naturally occurring cell death, by signalling through p75 and Trk receptors. In the Drosophila optic lobe, a wave of apoptosis occurs during neural circuit development; however, whether this also involves neurotrophism remains unresolved. Drosophila neurotrophins (DNTs) are encoded by spätzle (spz) paralogue genes and bind Toll receptors instead. Here, we focused on DNT-3 (previously known as spz-3) and DNT-2 (also known as spz-5 ) to ask whether they underlie neurotrophism in visual system development. We show that DNT-3 ( spz-3 ) and DNT-2 ( spz-5 ) are both expressed in the retina and in medulla neurons, and multiple Tolls are expressed across lamina and medulla neurons. Over-expression of DNT-3 ( spz-3 ) and DNT-2 ( spz-5 ) could rescue natural occurring cell death, whereas their loss of function caused cell death, showing that DNT-3 and DNT-2 can, and are required to, promote cell survival during optic lobe development. Importantly, DNT-2 is expressed in Mi1 neurons and Toll-2 in connecting L1 neurons. We show that DNT-2 functions in concert with Toll-2, as Toll-2 RNAi knock-down prevented the rescue of apoptosis by DNT-2 over-expression and all Toll-2+ neurons were lost in DNT-2 mutants. Furthermore, alterations in DNT-2 or Toll-2 expression levels impaired connectivity of L1 neurons at the M1 medulla layer and altered dendritic morphology of L1 neurons. These data suggest that L1 neurons could take up DNT-2 secreted from medulla neurons during the establishment of connectivity patterns. As DNT-3 ( spz-3 ) and DNT-2 ( spz-5 ) are expressed in the medulla and they could influence both lamina and medulla neurons, this suggests that their function maintaining cell survival could enable the stabilisation or alignment of connected neurons across medulla columns.

ABSTRACT Doyne honeycomb retinal dystrophy is an incurable juvenile macular dystrophy that leads to visual impairment by early to mid-adulthood. It is an autosomal dominant disorder caused by a c.1033T>C, p.Arg345Trp variant in EFEMP1 , and is characterised by the early onset extracellular deposition of drusen between the retinal pigment epithelium basement membrane and underlying layers of Bruch’s membrane. In this study, we developed an antisense oligonucleotide approach to target EFEMP1 . We reprogrammed patient-derived renal epithelial cells to induced pluripotent stem cells followed by directed differentiation to retinal pigment epithelium and compared the phenotype to gene-corrected and EFEMP1 knockout patient-derived retinal pigment epithelium. In the patient-derived disease model, remodelling of the extracellular matrix occurred with progressive accumulation of extracellular deposits containing the drusen-associated proteins apolipoprotein E and collagen IV, in addition to EFEMP1. Moreover, the intracellular accumulation of neutral lipids was evident. We developed an allele-specific antisense oligonucleotide which specifically and effectively promoted the clearance of the EFEMP1 c.1033T>C transcript in the patient-derived disease model following assisted or gymnotic delivery. In this disease model, gymnotic delivery led to a decrease in extracellular deposits and cleared the intracellular accumulation of lipids, even after the onset of this disease phenotype, suggesting this could be a practical and effective therapeutic approach.

Development of scalable and highly adaptable platforms to characterise high consequence infections are essential for limiting the impact of future viruses with pandemic potential. As part of this, an understanding of the virus-host interactions is vital, with the cell entry mechanism being crucial for the development of therapeutics and vaccines. Current approaches in this field depend on assays with authentic (live) viruses that require high containment facilities. However, these are hindered by high costs, need for highly trained staff, slow processing times and limited scalability. Using pseudotyped viruses (PV) expressing the chikungunya (CHIKV) envelope protein as a proof-of-principle, we developed a novel screening platform, Ceudovitox , to identify cellular factors involved in viral entry. PVs were engineered to express the herpes simplex virus-1 thymidine kinase, which following addition of ganciclovir, can selectively kill infected cells. Then a heterogenous pool of knockout cells were produced using the CRISPR-Cas9 library. Infection of these cells with the “killer” PV system permitted positive selection of cells refractory to viral infection and, through next generation sequencing, identification of a number of factors involved in CHIKV entry. Matrix metalloproteinases were identified as novel entry factors and demonstrated that MMP-targeting drugs efficiently inhibit PV and authentic CHIKV infections. These results suggest this platform holds great promise as a pandemic-preparedness tool that increases the capacity and speed of screening for cell entry factors. It is also a safe platform able to dissect the gene network involved in virus entry. Ceudovitox will help identify new therapeutic targets, thereby aiding the development of treatments against future outbreaks or pandemic pathogens and making a significant contribution to the “100 days” mission. Author Summary The increased threat of the emergence of new viral pathogens with pandemic potential highlights the importance of developing scalable high-throughput screening platforms to quickly characterise new emergent viral pathogens. Improving understanding of virus-host interactions, including how viruses enter cells, can help fast development of new targeted therapeutics and reduce pandemic burdens. Current approaches rely on using ‘live’ viruses, which are highly infectious and must be manipulated in high containment facilities, which are slow and cumbersome. Our new screening platform, Ceudovitox , is a safer, faster and more scalable alternative. Through using pseudotyped viruses, which can only undergo one replication cycle, we are able to distinguish virus entry factors of high containment viruses within highly abundant low containment facilities, allowing us to quickly characterise potential entry factors of high containment viruses. Within this study, Ceudovitox was shown to recognise both known and novel entry factors of Chikungunya virus; a virus which causes chronic arthralgia and arthritis. Matrix metalloproteinases were amongst novel entry factors identified and were seen to reduce virus infection in both pseudotyped and authentic virus assays, demonstrating how Ceudovitox can correctly discover virus entry factors of ‘live’ high containment viruses. Ceudovitox’s utility as a screening platform for high containment viruses and new emerging pathogens, has the potential increase understanding and speed up the development of new therapeutics during pandemics, helping to reach the ‘100 Day Mission’ of pandemic preparedness.

Somatic mutations in ribosomal proteins (RPs), including RPL5, have been reported in approximately 10% of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). In cancer, the incorporation of mutant RPs into ribosomes often disrupts canonical ribosome function, thereby contributing to disease development. In this study, we aimed to characterize the effects of the RPL5-I60V mutation in the context of T-ALL, focusing on its impact on translation and cellular responses to a panel of compounds in vitro. Using CRISPR-Cas9, we generated a homozygous knock-in mutant in Jurkat cells and investigated its effects on ribosome biogenesis. We observed both quantitative and qualitative alterations in the production of the large ribosomal subunit. Ribosomes containing the mutant RPL5 protein exhibited intrinsically increased protein synthesis activity, which correlated with enhanced cellular proliferation. We then evaluated the response of these mutant cells to a panel of compounds targeting protein synthesis at various levels—including an MNK1 inhibitor, metformin, silvestrol, homoharringtonine, anisomycin, resveratrol, and hygromycin B—as well as cytarabine, a chemotherapeutic agent commonly used in T-ALL treatment. Our results showed that the RPL5-I60V mutation confers increased sensitivity to most of these compounds, with the exception of hygromycin B. This study advances our understanding of how oncoribosomes contribute to cancer pathogenesis and highlights the therapeutic potential of directly or indirectly targeting altered ribosomes, offering insights for the development of personalized treatment strategies. Highlights RPL5 mutations have been reported in T-ALL Ribosomes containing the RPL5 I60V mutation exhibit increased translational activity Increased proliferation is observed in cells harboring the RPL5-I60V mutation RPL5-I60V confers a specific drug sensitivity profile Statements and Declarations Competing interests: The authors declare that they have no conflict of interest.

Abstract Ribosomal DNA (rDNA) double-strand breaks (DSBs) threaten genome integrity due to the repetitive and transcriptionally active nature of rDNA. The nucleolus, while central to ribosome biogenesis, also functions as stress sensor. Here, we identify a transcription-dependent mechanism in which RNA polymerase II (RNAPII) is essential for homologous recombination (HR) repair of rDNA DSBs. Using CRISPR-induced breaks, high-resolution imaging, and transcriptional inhibition, we show that RNAPII activity drives the formation of nucleolar repair caps. Mechanistically, CtIP promotes RNAPII recruitment and H3K36 trimethylation at rDNA lesions, facilitating HR. Disruption of this RNAPII–CtIP–H3K36me3 axis impairs cap formation and repair, leading to persistent damage. RNAPII inhibition exacerbates genome instability and synergizes with rDNA breaks to induce cancer cell death, without acutely impairing ribosome function. These findings uncover a co-transcriptional mechanism of rDNA repair and highlight RNAPII-mediated chromatin remodeling and spatial reorganization as key to nucleolar genome maintenance and potential targets for cancer therapy.

Summary Emerging research has implicated Alzheimer’s disease (AD) pathology with dysregulation of many key pathways in microglia, including lipid transport and metabolism, phagocytosis of plaques, and lysosomal function. However, the exact mechanisms underlying these pathways remain poorly understood. Leveraging high-throughput CRISPR screens to understand the interplay between these pathways may enable novel therapeutic strategies for AD and other neurological diseases. Here, we constructed activation and interference CRISPRa/i libraries targeting 203 genes, 71 of which were identified through neurodegenerative GWAS, and 132 additional genes linked to microglial functions. We used this library to conduct pooled CRISPRa/i screens across a range of functional assays relating to lipid metabolism and lysosomal function using a monocytic cell line, THP-1. We identified a core set of lipid and lysosome mediators and validated a subset in primary macrophages. To gain insights into transcriptional states modulated by these genes we also applied the CRISPRa/i libraries to Perturb-seq, enabling us to capture transcriptomic changes. Through non-negative matrix factorization, we identified five gene programs altered by our perturbation library. We then used an integrative analysis of functional screen data with Perturb-seq data that enabled us to uncover novel functions and genetic relationships between perturbations. This multidimensional resource links genetic perturbations to phenotypes and transcriptional programs, establishing a scalable framework for systematic gene discovery in neurodegeneration and beyond. Abstract Figure Graphical Abstract

Allelic variation can impact viral clearance and disease severity, but the effect of the autoimmunity-associated allelic variant of Ptpn22 (PEP-R619W) on antiviral immunity remains incomplete. This research defines how the loss of Ptpn22 (PEP-null) and PEP-R619W changes antiviral immunity during acute coronavirus infection. We address the hypothesis that CRISPR/Cas9-generated PEP-null and PEP-R619W mice have enhanced antiviral immunity over PEP-WT mice during coronavirus infection. Following Mouse Hepatitis Virus (MHV) A59 infection, we interrogated pathology, cytokine production, and cellular responses in the blood, spleen, and liver of PEP-WT, PEP-null, and PEP-R619W mice. Key findings show that PEP-R619W mice have reduced viral titer and weight loss, increased survival, and more mature NK cells in the liver and spleen compared to PEP-WT mice. Interestingly, protection against disease in PEP-null mice was inoculation-dose-dependent, whereas PEP-R619W conferred immunity regardless of infection dose. Further, Rag1-/- PEP-R619W mice had increased survival and reduced viral titer over Rag1-/- PEP-WT mice. PEP-R619W mice also had higher concentrations of IFNγ and enhanced IFNγ production by mature NK cells in the liver at 3 days post-infection. Finally, NK cell depletion elevated PEP-R619W viral titer to similar levels as PEP-WT mice. This is one of the first studies investigating the role of Ptpn22 within NK cells and demonstrates that the Ptpn22 allelic variant augments NK cell function and is beneficial during coronavirus infection. One Sentence Summary The Ptpn22 autoimmunity-associated allele uniquely augments innate immunity to protect against acute coronavirus infection.

Tissue-resident macrophages efficiently internalize Aspergillus fumigatus spores, forming a critical first line of defense against infection. However, the mechanisms that these cells use to control spores in vivo remain incompletely defined. Here, we used the live imaging capabilities of the larval zebrafish host model to assess the role of the v-ATPase complex in macrophage-mediated defense against A. fumigatus in a whole vertebrate animal. For the first time we are able to visualize co-localization of A. fumigatus spores with the key v-ATPase subunit Atp6v1h in macrophages inside of an infected animal. As macrophages only have a low ability to kill spores, this co-localization occurs as early as 1-day post-injection and persists for multiple days. Surprisingly, macrophage spore killing is not further reduced by targeting of atp6v1h with CRISPR/Cas9. Instead, v-ATPase deficiency profoundly impacts macrophage-mediated control of spore swelling, decoupling the macrophage functions of spore killing and inhibition of germination. We also identify a role for the v-ATPase complex in macrophage control of extracellular hyphal growth. These effects on macrophage function drive significantly decreased host survival in larvae lacking a functional v-ATPase. We also report broad effects of v-ATPase deficiency on macrophage numbers, apoptosis in the hematopoietic tissue, and potential neutrophil functions, reflecting the importance of this complex in host antifungal immunity. Graphical Abstract Highlights v-ATPase function in macrophages restricts Aspergillus germination and hyphal growth Spore killing by macrophages is independent of v-ATPase function Loss of macrophage v-ATPase mimics the effects of macrophage deficiency in vivo

The transcription factor ATF6 has a central role in adapting mammalian cells to endoplasmic reticulum (ER) stress via the Unfolded Protein Response (UPR). This has driven efforts to identify modulators of ATF6 signalling. Here, an unbiased genome-wide CRISPR-Cas9 screen performed in Chinese Hamster Ovary (CHO) cells revealed that proteolytic processing of the ATF6α precursor to its active form was impaired in CHO cells lacking the ER-resident solute carrier SLC33A1, a transporter involved in acetyl-CoA import, sialylation and Nε-lysine protein acetylation. Cells lacking SLC33A1 constitutively trafficked the ATF6α precursor to the Golgi, but exhibit impaired subsequent Golgi processing, correlating with altered ATF6α Golgi glycosylation. SLC33A1 deficiency also deregulated activation of the IRE1 branch of the UPR, pointing to a selective loss of ATF6α-mediated negative feedback in the UPR. Notably, Slc33a1 -deleted cells accumulated higher levels of unmodified sialylated N-glycans, precursors to acetylated glycans, likely reflecting impaired glycan processing. By contrast, deletion of ER-localised acetyltransferases NAT8 and NAT8B, which catalyse protein Nε-lysine acetylation in the secretory pathway, did not replicate the ATF6α processing defects observed in Slc33a1 -deficient cells. Together, our findings highlight a role for SLC33A1-mediated metabolite transport in the post-ER maturation of ATF6α and point direct links between small-molecule metabolism and branch-specific signalling in the UPR.

ABSTRACT Tomato is one of the most produced and consumed vegetables globally due to its nutritional benefits, sensory characteristics, and cultural importance. However, tomato fruit has a short shelf-life, which can be extended by postharvest techniques, but often at the expense of fruit quality, leading to consumer dissatisfaction. To address this challenge, we modified the upstream regulatory regions of Ripening inhibitor (RIN), a master regulator of tomato fruit ripening, utilizing a CRISPR/Cas9 multiplex system. This approach enabled the creation of a population of tomato fruit with mutations of varying severity, which could have far-reaching effects on the RIN-induced gene regulatory network in tomato fruit, leading to downstream changes in fruit traits. We have generated 264 first-generation (T 0 ) transgenic lines of RIN promoter mutants with diverse genetic lesions and RIN transcriptional levels. Our study revealed a non-linear relationship between promoter mutations and gene expression, highlighting the potential roles of certain types of mutations in regulating RIN transcription. Future work will focus on evaluating fruit traits from mutants with pronounced changes in RIN expression, as well as performing transcriptomic analysis to explore the mechanisms underlying fruit quality modifications due to genome editing.

ABSTRACT Alveolar rhabdomyosarcoma (aRMS) is a fusion-driven pediatric cancer with poor survival and limited therapeutic options. To uncover novel vulnerabilities, we employed complex-based analysis of the DepMap functional genomic data, identifying CDK8 as a dependency in aRMS. Both CDK8 knockout and pharmacologic inhibition impaired tumor cell growth and induced myogenic differentiation in vitro and in vivo . Compared to genetic loss, CDK8 inhibition induced more dynamic transcriptional changes. With a genome-scale CRISPR-Cas9 drug modifier screen, we determined that the maximal anti-tumor activity of the CDK8 inhibitor requires the presence of the Mediator kinase module and transcriptional cooperation with the SAGA complex. We further identified SIX4 as a key transcription factor mediating CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes and tumor cell proliferation. These findings suggest a distinct gain-of-function mechanism of the CDK8 inhibitor and establish a strong rationale for CDK8 inhibition as a differentiation-inducing therapeutic strategy in aRMS. STATEMENT OF SIGNIFICANCE We provide a framework for uncovering therapeutic targets by network-based analysis of functional genomic screens. We identify CDK8 as a druggable target in aRMS and determine that CDK8 inhibition drives myogenic differentiation and impairs tumor progression via a collaborative mechanism involving the Mediator kinase module, SAGA complex, and SIX4.

ABSTRACT Mitochondrial complex I (CI) deficiency represents a common biochemical pathophysiology underlying Leigh syndrome spectrum (LSS), manifesting with progressive multi-system dysfunction, lactic acidemia, and early mortality. To facilitate mechanistic studies and rigorous screening of therapeutic candidates for CI deficient LSS, we used CRISPR/Cas9 to generate an ndufs2 -/- 16 bp deletion zebrafish strain . ndufs2 -/- larvae exhibit markedly reduced survival, severe neuromuscular dysfunction including impaired swimming capacity, multiple morphologic malformations, reduced growth, hepatomegaly, uninflated swim bladder, yolk retention, small intestines, and small eyes and pupils with abnormal retinal ganglion cell layer. Transcriptome profiling of ndufs2 -/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Similar transcriptomic profiles were observed in ndufs2 -/- missense mutant C. elegans ( gas-1(fc21) ) and two human CI-disease fibroblast cell lines stressed in galactose media. ndufs2 -/- zebrafish had 80% reduced CI enzyme activity. Unbiased metabolomic profiling showed increased lactate, TCA cycle intermediates, and acyl-carnitine species. One-carbon metabolism associated pathway alterations appear to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in ndufs2 -/- larvae. Overall, ndufs2 -/- zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.

ABSTRACT Neuroblastoma is a childhood cancer, arising in the developing sympathetic nervous system. Differentiation therapy with 13-cis-retinoic acid (RA) is routinely given to children with high-risk neuroblastoma in the minimal residual disease setting to prevent relapse, however there is little understanding of which patients benefit from RA therapy. ATRX alterations are identified in 10% of high-risk neuroblastomas and associated with poor outcomes. The commonest type of ATRX alterations in neuroblastoma are in-frame multi-exon deletions, followed by nonsense mutations predicted to result in loss-of-function ( ATRX LoF). We treated paired ATRX wild-type and ATRX LoF neuroblastoma cell lines with RA and show that cells with ATRX LoF fail to upregulate direct RA target genes. Cells with ATRX LoF also show reduced chromatin accessibility at genes involved in differentiation and development following RA treatment. Conversely, neuroblastoma models with in-frame deletions mount a response to RA and show in-vitro sensitivity to RA. Taken together this shows that the mechanism of differentiation in ATRX -altered neuroblastoma depends on the type of ATRX alteration, with implications relating to both oncogenesis and therapeutic response.

Dravet syndrome (DS) is a severe childhood genetic epilepsy, caused by de novo heterozygous mutations in the SCN1A gene, resulting in a loss of function of the voltage-gated sodium ion channel, Nav1.1. Nav1.1 is expressed in the brain and at a lower level, in the heart. The disease manifests in the first year of life. Patients exhibit tonic-clonic seizures, febrile seizures, cognitive decline, development delays, ataxia, and in some cases sudden unexpected death from epilepsy (SUDEP). Here we have developed a novel AAV-F mediated CRISPR-Cas-inspired RNA targeting system (CIRTS) pre-clinical treatment to increase endogenous Scn1a to ameliorate the disease phenotype in a clinically relevant heterozygous loss of function mouse model of DS. We designed novel guide RNAs (gRNAs) to target the long non-coding RNA, (or natural antisense transcript) of Scn1a to increase the expression of Scn1a mRNA in DS mice. We show that intracerebroventricular and intravenous administration of AAV-F-CIRTS-gRNA9 to target the brain and the heart to neonatal Scn1a+/- mice resulted significant increase in survival and a reduction in SUDEP and spontaneous seizures. Furthermore, we have shown increased expression of endogenous Scn1a in treated AAV-F-CIRTS-gRNA9 Scn1a+/- mice. These findings provide proof of concept evidence that an AAV-F-CIRTS mediated therapy hold promise as a potential long-term treatment for DS.

2 2.1 Background Adipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. 2.2 Methods Small RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. 2.3 Results Both cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. 2.4 Conclusions These results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.

Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary e\ectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo . Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.

Radiotherapy is part of the standard-of-care for glioblastoma, yet tumors invariably recur as incurable lesions post-treatment. Recent studies suggest that radiation-induced astrocyte reactivity fosters a tumor-supportive environment, however effective strategies targeting reactive astrocyte phenotypes are lacking. Using a novel image-based assay, we screened over 1,700 small molecule compounds, identifying 29 that inhibit radiation-induced astrocyte reactivity in human astrocytes. Among these, Flunarizine, a calcium-entry blocker approved for migraine treatment, significantly reduced astrocyte reactivity in vitro and in vivo. In a genetically engineered glioblastoma mouse model, combining Flunarizine with radiotherapy markedly improved survival without affecting unirradiated controls, indicating specificity for a radiation-induced phenotype. Mechanistically, Flunarizine inhibited radiation-induced fibrosis in vivo and directly suppressed astrocytic TGF-beta activation in vitro. Notably, Flunarizine treatment had no direct effect on primary glioblastoma cells, emphasizing its microenvironmental specificity. In conclusion, we identified Flunarizine as a promising repurposed compound capable of effectively mitigating radiation-induced astrocyte reactivity and delaying glioblastoma recurrence. This approach offers a viable therapeutic strategy to enhance current glioblastoma treatments. Graphical abstract

Emerging evidence suggests that chaperones not only facilitate the folding of nascent polypeptides but also interact with quasi-native or natively folded proteins. However, the biological significance of these interactions remains unclear. In our previous study, we demonstrated that the molecular chaperone-like protein BSA reshapes the energy landscape of folded client proteins, increasing the population of their excited states. Building on this, we further explored chaperone-enzyme interactions and found that chaperones Spy and Hsp70 affected the conformational exchanges of folded lysozyme, leading to increased catalytic activity. Additionally, we observed that the influence of chaperones on enzyme activity extended to other chaperone-enzyme interactions. Molecular chaperones Hsp70, Spy, Hsp104, and Hsp20, enhanced the catalytic activity of diverse enzymes, such as alkaline phosphatase (PHPT1), DNA polymerase from Pyrococcus furiosus ( Pfu pol), endonuclease CRISPR-Cas12a/Cas13a, and xylanase. Our findings reveal an unexpected role of chaperones in modulating the activity of folded enzymes, broadening the known functions and mechanisms of molecular chaperones in biological systems. Furthermore, these insights highlight the potential applications of chaperones in enzyme-based biological assays, diagnostics, and biomanufacturing, as well as studying of new roles of upregulated chaperones in systemically modulating cellular biological processes.

ABSTRACT Ultraviolet-B radiation (UV-B) poses a major challenge to all forms of plant life. The liverwort Marchantia polymorpha (Marchantia) serves as a key model organism to study signaling pathways and to infer their evolution throughout the green lineage. Marchantia expresses key components of UV-B signaling, including the photoreceptor UV RESISTANCE LOCUS 8 (MpUVR8), the WD40-repeat protein REPRESSOR OF UV-B PHOTOMORPHOGENESIS (MpRUP), the E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC 1 / SUPPRESSOR OF phyA-105 (MpCOP1/MpSPA), and the transcriptional regulator ELONGATED HYPOCOTYL 5 (MpHY5). Here, we show that MpUVR8 exists as a homodimer in its ground-state in vivo, then monomerizes and accumulates in the nucleus upon UV-B activation. Activated MpUVR8 interacts with MpCOP1, triggering growth inhibition, gene expression changes, biosynthesis of UV-absorbing metabolites, and photoprotection, which overall contributes to UV-B stress tolerance. MpRUP facilitates redimerization of MpUVR8 and Mp rup null mutants show enhanced UV-B photomorphogenesis, indicating that MpRUP efficiently represses MpUVR8 signaling. Unlike the case in Arabidopsis and in contrast to the strong Mp cop1 mutant phenotype, Mp spa mutants develop only a weak constitutive photomorphogenesis phenotype, indicating that COP1 function is more independent of SPA in Marchantia than in Arabidopsis. Interestingly, however, Mp spa is linked with a hyper-responsive UV-B phenotype, suggesting that MpSPA is a negative regulator of MpUVR8 signaling. Our findings demonstrate that core components of UV-B signaling existed in the last common ancestor of extant land plants; however, regulatory interactions have diversified in different lineages since their divergence more than 400 million years ago.

CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed isoform-specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 48.3% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.

T-cell-based therapies have gained momentum in recent decades, revolutionizing treatment options for cancer patients. Currently, CRISPR Cas-9 and RNAi technologies are commonly employed for understanding genetic networks in primary T cells. These technologies involve mainly electroporation or viral vectors to deliver the cargo required for gene editing or silencing and they pose some limitations such as reduced cell viability, poor transfection efficiency, and insertional mutagenesis. Dharmacon™ has developed chemically modified self-delivering siRNAs termed Accell™ siRNAs with wide cell type applicability transcending the need for transfection reagents or viral vectors for intracellular siRNA delivery. In this study, using human primary CD4-T cells as a model, we optimized Accell™ siRNA uptake and gene expression knockdown in unstimulated T cells and optimized a workflow to efficiently knockdown gene expression in primary T cells without compromising cell viability. This method is useful for rapidly investigating gene functions in primary T cells in a resting state.