Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms—particularly those involving phosphorylation and deacetylation of specific histone residues—have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

Phage defense systems in bacteria exhibit high degrees of modularity, with sensing, signal transmission, and effector enzymes frequently being exchanged among phage defense gene clusters. In this study, we capitalized on this modularity to discover phage defense systems by searching for defense-associated modules in new gene contexts. This approach revealed a large and interconnected network of modular components distributed across diverse gene clusters. From over 500 candidate defense systems, we selected nine for experimental testing and validated three: Dionysus, a TerB-encoding system that disrupts early phage infection vesicle formation by Jumbo phages; Ophion, a Radical SAM-containing system that prevents the formation of the Jumbo phage nucleus; and Ambrosia, a tightly regulated RM-like system. Collectively, we demonstrate that leveraging the modular architecture of phage defense systems is an effective approach to their discovery.

ABSTRACT Self-renewal and differentiation are at the basis of hematopoiesis. While it is known that tight regulation of translation is vital for hematopoietic stem cells’ (HSCs) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by ribosomal RNA (rRNA) methylation dynamics. Using ultra-low input ribosome-profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency changes progressively with differentiation and can distinguish between discrete cell populations as well as to define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification - 2’-O-methyl (2’OMe). We found that, like translation efficiency, 2’OMe dynamics followed a distinct trajectory during erythroid differentiation. Genetic perturbation of individual 2’OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSCs with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex-vivo , and loss of fitness in-vivo in bone marrow transplantations. Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2’OMe profiles during differentiation of non-hematopoietic stem cells. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation. KEY POINTS Hematopoietic differentiation is associated with rRNA methylation dynamics to control cell-type-specific translation. Translation efficiency can distinguish discrete cell types and define differentiation trajectories. HSC fitness is regulated by a single rRNA methylation.

Background The advent of CRISPR-Cas9 genome editing has brought about a paradigm shift in molecular biology and gene therapy. However, the persistent challenge of off-target effects continues to hinder its therapeutic applications. Unintended genomic alterations can lead to significant genomic damage, thereby compromising the safety and efficacy of CRISPR-based therapies. Although in-silico prediction tools have made substantial progress, they are not sufficient for capturing the complexity of genomic alterations and experimental validation remains crucial for accurate identification and quantification of off-target effects. In this context, Genome-wide Unbiased Identification of Double-strand breaks Enabled by Sequencing (GUIDE-Seq) has emerged as a gold standard method for the experimental detection of off-target sites and assessment of their prevalence by introducing short double-stranded oligonucleotides (dsODNs) at the break sites created by the nuclease. The bioinformatic analysis of GUIDE-Seq data plays a pivotal yet challenging role in accurately mapping and interpreting editing sites and current pipelines suffer limitations we aim to address in this work. Results In this study, we present a rapid and versatile single-command pipeline designed for the comprehensive analysis of GuideSeq and similar techniques of sequencing. Our pipeline is capable of simultaneously processing multiplexed libraries from different organisms, PCR orientations, and Cas with different PAM specificities in a single run, all based on user-specified sample information. To ensure reproducibility, the pipeline operates within a closed environment and incorporates a suite of well-established bioinformatics tools. Key novel features include the ability to manage bulges in gDNA/gRNA interaction and multi-hit reads, and a built-in tool for off-target site prediction. The pipeline generates a detailed report that consolidates quality control metrics and provides a curated list of off-target candidates along with their corresponding gRNA alignments. Conclusions Our pipeline has been tested and successfully applied to analyze samples under a variety of experimental conditions, including different source organisms, PAM motifs, dsODN sequences and PCR orientations. The robustness and flexibility of our pipeline make it a valuable tool for researchers in the field of genome editing. The source code and comprehensive documentation are freely accessible on our GitHub repository: https://github.com/gcorre/GNT_GuideSeq .

The CRISPR/Cas9 system deployed through crosses of transgenic lines expressing Cas9 and gRNA facilitates efficient mutagenesis. However, its application in non-model insects remains limited, primarily due to a lack of well-characterized promoters capable of driving robust and stable expression of Cas9 and gRNA . In the malaria mosquito Anopheles sinensis , we evaluated several ovary-biased promoters— Asvasa2, Aszpg , and Asnanos —for driving Cas9 expression. Notably, the Asvasa2 promoter mediated mutagenesis in nearly 60% of G 0 individuals following microinjection of gRNA Aswhite . Among four RNA polymerase III promoters derived from AsU6 genes, AsU6 -1 yielded the highest gRNA transcriptional output, enabling 62% editing efficiency in G 0 offspring. In addition, hybrid crosses between established transgenic lines demonstrated that the Asvasa2-Cas9 and AsU6 -1- gRNA combination enabled complete germline editing penetrance, where all F 2 progeny inherited the intended mutations. This work provides a essential genetic toolkit for synthetic biology applications in Anopheles mosquitoes and a scalable framework for engineering other non-model insects.

Summary Humans and animals are ubiquitously colonized by Enterobacteriaceae , a bacterial family that contains both commensals and clinically significant pathogens. Here, we report Enterobacteriaceae megaplasmids of up to 1.58 Mbp in length in infant and adult guts, and other microbiomes. Of 19 complete plasmid genomes, one was reconstructed from an E. coli isolate; others were linked to species of Citrobacter and Enterobacter via analysis of genome modification patterns. The detection of related plasmids in different Enterobacteriaceae , conjugation machinery, and more diverse modified motifs in certain plasmids compared to hosts suggests that these elements are self-transmissible, with a broad host range. The plasmids encode multi-drug efflux systems and potential secreted effectors. Up to 208 tRNAs are encoded and include sequence variants that may counter tRNA-centric defense mechanisms. Overall, the vast megaplasmid coding capacity may broaden host range, increase competitiveness, control invasion by other elements, and counter programmed cell death.

Abstract Secondary lymphoid tissues develop specialized reticular networks to facilitate immune cell communication and efficient activation of adaptive immunity. This stromal network architecture is robust, maintaining topology throughout extensive remodelling and tissue expansion in response to immune challenge. We have previously reported that cytoskeletal mechanics of the fibroblastic reticular cell (FRC) networks determine tissue tension, and that increased tension initiates stromal proliferation for lymph node growth. However, it is not known how FRCs maintain stromal network connectivity and what cellular mechanisms reinforce stromal cell-cell and cell-matrix interactions. Here, we present a signalling mechanism which coordinates reduced FRC contractility and induction of stromal cell protrusions. RhoA/C GTPase activity is blocked in FRCs to inhibit actomyosin contractility through contact with dendritic cells (DCs) and binding between podoplanin and the C-type lectin CLEC-2. We now find that an additional Rho GTPase target, the PKC family kinase PKN2, regulates the function of myristoylated alanine-rich protein kinase C-substrate (MARCKS). FRCs generate cell protrusions via MARCKS in response to DC contact, which reinforces stromal cell connectivity. In vivo, we found that PKN2 knock-out lymph nodes are unable to regulate MARCKS and show severely disrupted stromal architecture. These results reveal a mechanism of stromal/immune cell crosstalk which actively induces stromal protrusions – an essential component of lymph node remodelling to maintain tissue integrity during an adaptive immune response.

The intestinal mucus layer is essential for the integrity of the intestinal barrier. It is produced by goblet cells, whose depletion is common in colonic inflammation but remains poorly understood. Here, we show that goblet cell survival relies on a reciprocal dependence with newly discovered BEST4/CA7 + cells. We developed a method to follow BEST4/CA7 + and goblet cells in time from birth to death in human colon organoids. Notably, goblet cells induce BEST4/CA7 + fates in sister cells and other neighbors, using DLL1-mediated lateral activation of Notch-signaling. BEST4/CA7 + cells in turn promote goblet survival, with the latter depleting rapidly after differentiation in absence of BEST4/CA7 + cells. This apoptosis inhibition does not require direct cell-cell contact and instead depends on their shared lumen. Such differentiation and survival interdependencies may be relevant beyond the maintenance of mucosal homeostasis.

ABSTRACT Agroinfiltration of Nicotiana benthamiana is frequently used to produce recombinant proteins, both for plant science and for molecular pharming. Here, we introduce two genome-edited lines of N. benthamiana lacking two polyphenol oxidases (PPOs). These double ppo knockout lines grow slightly faster than wild-type and show similar levels of transient GFP expression. However, leaf extracts produced in native buffers stay greener and show much less native crosslinking of Rubisco and other proteins, demonstrating that PPO depletion reduces enzymatic browning and protein crosslinking in leaf extracts. Transient PPO1 expression in the ppo mutant restores browning and crosslinking in leaf extracts. These ppo mutants offer tremendous opportunities to increase yield and purity of recombinant proteins and study protein complexes, as illustrated with a nearly 4-fold increase in purification yield and a substantial improvement of protein purity upon purification of transiently expressed His-tagged tomato immune protease P69B from total leaf extracts.

ABSTRACT Imps are a highly conserved family of RNA-binding proteins involved in embryonic development, cancer progression, and neurogenesis. However, the molecular pathways and RNAs regulated by Imp to control these processes remain poorly understood. Embryos derived from Imp mutant germline clones arrest development, and transcriptome analysis revealed significant dysregulation of genes involved in cell growth, differentiation, tube morphogenesis, neuronal projection development, and RNA metabolism, along with de-repression of transposable element (TE) RNAs. Consistent with these findings, Imp mutant embryos display TE-overexpression phenotypes, are smaller in size, and exhibit defective organ development, including impaired tracheal branching and gastrulation. Reduced levels of Imp at the larval neuromuscular junction (NMJ) impair synaptic bouton formation and decrease adult longevity. RIP-seq experiments showed that Imp-associated RNAs are enriched for TE RNAs. Proteomic analyses confirmed that several TE-encoded proteins are upregulated in Imp mutant embryos. Specifically, the Ty1 family retrotransposon Copia was derepressed. Consistent with recent findings that Copia is a potent inhibitor of synaptogenesis, its upregulation likely contributes to the impaired NMJ formation and broader embryonic defects observed in Imp mutants. Moreover, Imp associates with piRNA pathway proteins, ensures Piwi nuclear localization, and—like piwi mutants—its loss disrupts TE silencing and causes position-effect variegation (PEV) defects. The analysis of Imp complexes further points to potential mechanisms by which Imp may regulate TE expression. Overall, these results indicate that Imp maintains genome stability and ensures proper developmental progression and neuronal activity by regulating post-transcriptional processes and suppressing transposons.

ABSTRACT Multidrug transporters, including multidrug resistance-1 (MDR1), are recognized chiefly for effluxing chemotherapeutic drugs out of tumor cells. However, they are also expressed in many normal cells and tissues, including lymphocytes, but their physiological role is less well-understood. Here, we investigated the role of MDR1 in tumor-specific CD8 T cells (TST), which are critical in antitumor immunity and key targets of immunotherapies. Using a clinically-relevant genetic liver cancer mouse model, we investigated the efflux dynamics of TST as they underwent activation, proliferation, and differentiation to dysfunctional states in tumor-bearing hosts. Surprisingly, we found that late-stage/terminally dysfunctional TST had the highest efflux capacity in both murine and human liver tumors. TST upregulated transcription of Abcb1a , encoding MDR1. We used CRISPR/Cas9 to generate MDR1-deficient TST, which persisted poorly in tumor-bearing mice as compared to MDR1-sufficient TST. MDR1 expression improved TST viability and reduced reactive oxygen species accumulation. Loss of MDR1 made T cells more susceptible to cytotoxic chemotherapy-induced cell death. Our findings demonstrate a role for MDR1 in regulating TST persistence and oxidative stress, with implications for antitumor T cell therapies in patients and immune regulation following cytotoxic chemotherapy.

Nitric oxide (NO) is an important signaling molecule in flowering plant immunity. It rapidly accumulates in response to pathogen perception. In addition to it’s direct response to microbes, NO controls a range of defence responses primarily through S -nitrosylation. This process is a redox-dependent modification where a NO group attaches to the thiol of a cysteine residue, creating an S -nitrosothiol (SNO). To explore the role of S -nitrosylation more broadly, we characterised the single-copy S - nitrosoglutathione reductase 1 (Mp GSNOR1 ) gene in the liverwort Marchantia polymorpha (Marchantia), a representative of a lineage widely diverged from flowering plants. We generated loss-of-function alleles using CRISPR/Cas9 genome editing. Disrupting Mp GSNOR1 resulted in pronounced morphological alterations, highlighting the role of GSNOR1 in the structural development of Marchantia. Additionally, we show that Mp GSNOR1 is essential for SNO homeostasis and immune function. Our results suggest that GSNOR was part of the tool kit of the ancestral land plant and functioned in immunity and development. Highlight First evidence from a Liverwort shows GSNOR controls immunity and development via S -nitrosylation, revealing these regulatory roles as ancient traits of land plants.

Cellular senescence is a hallmark of aging and a promising target for extending human healthspan. Senescence is often accompanied by upregulation of the key senescence marker gene CDKN2A , yet the mechanism underlying its transcriptional activation remains unclear due to complex cis -regulations within the 9p21.3 locus. Here, we performed complementary CRISPR activation and interference screens in human mesenchymal stromal cells (MSCs) to systematically map non-coding cis -regulatory elements (CREs) at this locus that epigenetically regulate senescence. This approach revealed senescence-regulating CREs (SenReg-CREs) that bidirectionally modulate senescence through P16 INK4a and P15 INK4b . Notably, we identified a primate-specific short interspersed nuclear element (SINE) MIR3 embedded within the most potent distal SenReg-CRE. Deletion of this SINE:MIR3 accelerated senescence, revealing its potential insulator function in restraining CDKN2A/CDKN2B activation. Collectively, these findings reveal novel mechanisms underlying senescence-associated transcriptional activation of CDKN2A/CDKN2B and demonstrate that senescence is malleable through manipulation of regulatory element activity, highlighting the potential of epigenetically targeting these SenReg-CREs for senomorphic interventions.

Summary The NLRP3 inflammasome is a major driver of immunopathology, making it a sought-after drug target. In spite of two decades of intense research, its precise activation mechanism remains elusive, impeding inhibitor design. NEK7 was reported as essential for NLRP3 activation, and several newly identified inhibitors were suggested to act by interfering with their interaction. Here we report that NEK7 accelerates, but is in principle dispensable for NLRP3 activation. The onset of inflammasome activation was unaltered in the absence of NEK7, yet the rate of cells to undergo inflammasome formation and subsequent pyroptosis was approximately 4-fold reduced. Therefore, therapeutic targeting of the NEK7-NLRP3 interaction might have an incomplete effect, which should be considered for drug development. We confirmed entrectinib as a NEK7-dependent inhibitor, while other published compounds turned out not to rely on it. Our results support two possible scenarios for the role of NEK7 in NLRP3 activation: either, NEK7 accelerates one unique pathway of NLRP3 activation, or it is essential for a fast pathway, while being dispensable for a second, slower mode of NLRP3 activation.

Quantitative cell biology often studies migration and the cell-cycle (CC) in separate assays, limiting mechanistic insights, particularly under geometric confinement. Here, we introduce a vertically integrated platform for simultaneously tracking single-cell migration and assessing CC under confinement. Our system integrates cell engineering via multiplexed sensors for cell-cycle, actin, and tubulin, as well as photopatterned engineered extracellular matrix (ECM) islands of defined sizes. It also features an automated, high-throughput pattern-aware imaging pipeline (Fab2Mic) that enables on-pattern, joint migration-CC assessment in the same live cells. Since the local microenvironment plays a critical role in metastasis by constraining cell behaviors within spatial boundaries, we used an HT1080 fibrosarcoma model as an illustrative case. Where static phenotyping yielded 40% G1 and 60% S/G2/M, with larger cell areas and tubulin spread in the S/G2/M phase, dynamic phenotyping via live-cell imaging confirmed CC-linked motility, with faster instantaneous velocities in G1, exemplifying the CC-migration correlations. These phenotypes were modulated by the spatial confinement imposed by the engineered ECM islands. Stronger confinement reduced cell area and tubulin spread and increased the frequency of abnormal CC events, particularly Long G1 states on smaller engineered ECM islands. It also induced a confinement-specific S/G2/M-G1 mitotic slippage, observed only under our confined conditions. Together, this vertically integrated system suggests that confinement may continuously tune migration–CC coupling and provides a deployable pipeline for CC-aware mechanobiology and screening. Moreover, we stress how dynamic imaging provides access to variables that are difficult or impossible to infer from static snapshots, including velocity and CC timing.

Genome editing is now available for many crops. It has increased our ability to study gene function and has changed the field of plant transgenesis. Nevertheless, the ability to regenerate plants from cell culture remains a limiting factor for many crops, and even for species with a good regeneration potential, some accessions remain recalcitrant. The physiological state of plant cells is involved in the process of plant growth and development and is closely linked to the network involving MAP-kinase signaling pathway. Some of the defense genes activated during the cellular repair process of transgenesis show high homologies with mammalian defense genes. We thus compared the percentage of transgenic plants obtained by CRISPR-Cas9 mutation in four genes involved in sugar and acid metabolism after supplementation with different mammalian growth factors and cytokines in six tomato accessions presenting a range of regeneration levels. We demonstrated, through three years of transgenesis experiments, that the use of mammalian growth factors during transgenesis improved regeneration rate of recalcitrant tomato accessions. We demonstrated that using cytokines not only improved transformation of difficult-to-transform accessions but also the production rate of stable secondary lines. Summary statement Supplementation of transformation medium with mammalian growth-regulating factors enhanced regeneration of tomato recalcitrant genotypes

2-Oxoglutarate-dependent Dioxygenases (2OGDDs) are a family of enzymes requiring molecular oxygen, 2-oxoglutarate, reduced iron, and ascorbic acid to function. This dependency renders them key sensors of the cell's metabolic state, driving crucial functions when oxygen or metabolic homeostasis is perturbed, including adaptation to low oxygen, epigenetic control of gene transcription, and the reshaping of metabolic pathways. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), a 2OGDD that alters epigenetic marks, is essential for DNA damage repair and is a key regulator of cell metabolism. Notably, JMJD5 is often lost in hepatocellular carcinoma, which correlates with poor overall survival. Despite its biological significance, the molecular functions of JMJD5 remain unresolved, and its physiological targets are elusive. Here, we identify and characterise a novel signalling pathway where JMJD5 hydroxylates an arginine residue on the protein ISY1. This modification enables ISY1 to bind to and reduce the activity of Protein Arginine N-methyltransferase 6 (PRMT6). Significantly, the inactivation of PRMT6 rescues the majority of the molecular phenotype driven by JMJD5 loss, establishing the JMJD5-ISY1-PRMT6 pathway as the principal executor of JMJD5's enzymatic function. This signalling pathway clarifies existing controversies regarding JMJD5's function and identifies PRMT6 as a potential therapeutic target for treating cancers that lack JMJD5.

Summary RH5-Interacting Protein (RIPR) is essential for the invasion of Plasmodium into host red blood cells and is currently being studied as a novel malaria vaccine candidate in Phase 1a clinical trials. To study the genetic diversity of RIPR, deep amplicon sequencing was used to identify RIPR mutations in Plasmodium falciparum clinical isolates (n=89) collected in Kédougou, a high malaria transmission region of Senegal. We identified nonsynonymous single nucleotide polymorphisms (SNPs) in 64/89 (71.9%) of the samples. In total, 26 non-synonymous SNPs were identified, of which 15 were novel. 16/26 SNPs were able to be threaded onto existing RIPR crystal structures to predict the effects of SNPs on RIPR stability. 7/16 mutations were predicted to destabilize RIPR while 2/16 increased the stability of RIPR. Additionally, we identified 3 SNPs (Q737K, T738K, V840L) in the EGF5-8 domains of RIPR where neutralizing antibodies are known to bind.

Precise cis -regulatory control of gene expression is essential for plant growth. In Arabidopsis thaliana , PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY) peptides and their receptors (PSYRs) mediate growth-stress trade-offs, yet the transcriptional regulation of these genes remains poorly understood. Here, we mapped transcription factor (TF)-promoter interactions for nine PSY and three PSYR genes by combining high-throughput enhanced yeast one-hybrid screening with DNA Affinity Purification sequencing (DAP-seq) data, uncovering 1,207 interactions that reveal both shared and gene-specific regulatory relationships, defining the global TF-promoter interaction network of the PSY / PSYR pathway. Functional analysis of 25 TF mutants identified 12 regulators that significantly influence root growth, most acting as repressors. Of these, CYTOKININ RESPONSE FACTOR 10 (CRF10) emerged as a strong growth inhibitor. We identified a CRF10-binding motif in the PSYR3 promoter using DAP-seq data and validated it using eY1H. This motif is also located in the last 3′ terminal exon of Topoisomerase 3A ( TOP3A ). Guided by these insights, we used CRISPR/Cas9-mediated promoter editing to delete a small region encompassing or flanking a functional TF-binding site (TFBS). Removal of this motif, or of its surrounding region, enhanced root growth, yielding variants that retained root length comparable to the crf10 mutant. Our results suggest that the observed root growth phenotype results either from disruption of the CRF10 binding motif or from the mutation in the TOP3A exon.

ABSTRACT Controlling Mycobacterium tuberculosis (Mtb) infection requires a precisely balanced host inflammatory response. Too little inflammation leads to uncontrolled bacterial growth but exacerbated inflammation, activated by mediators such as TNF and type I IFN, inhibits effective antibacterial responses. How these immunopathological states are established is unknown. Deeper understanding of the pathways elicited upon initial Mtb infection of the host macrophage may reveal vital regulatory mechanisms that govern the subsequent inflammatory environment and ultimate resolution of infection. To elucidate these early regulators of inflammation, we performed a genome-wide CRISPR knockout screen in macrophages to identify genes that influence the induction of TNF and iNOS upon infection with Mtb. The resulting dataset is a valuable resource that includes genes representing a wide range of unexpected regulatory mechanisms that control cytokine responses to Mtb and also cell-intrinsic resistance to infection by the bacterial pathogen Listeria monocytogenes. We show that type I IFN signaling enhances TNF production early after infection, and IRF2 acts to inhibit induction of the antibacterial state of macrophages. Our data support a model in which early production of type I IFN in response to bacterial infection serves to increase innate antibacterial resistance during the earliest stages of infection.

Invasive fungal respiratory infections (IFRIs) remain a major cause of morbidity and mortality among immunocompromised patients, yet diagnosis continues to be hindered by nonspecific clinical features, limited sample accessibility, and the poor sensitivity or specificity of conventional tests. Microfluidic and microelectromechanical systems (MEMS)-based biosensing platforms have emerged as promising alternatives, enabling rapid, minimally invasive, and highly specific detection of fungal pathogens and host responses. Microfluidic nucleic acid and antigen assays allow on-chip amplification and immunodetection with reduced sample volumes and turnaround times, while CRISPR-enhanced systems further improve analytical sensitivity. Parallel advances in host-response profiling—including transcriptomic, proteomic, and cytokine-based signatures—have demonstrated feasibility for integration into lab-on-a-chip platforms. MEMS-based technologies extend this potential by facilitating real-time analysis of exhaled volatile organic compounds, mechanical biosensing of fungal DNA and antigens, and in situ monitoring of device-associated biofilms. Translational studies highlight potential applications across intensive care, hematology–oncology, and transplant settings, as well as in outpatient monitoring of high-risk populations. However, several challenges remain, including limited multicenter validation, matrix-related biofouling effects, and lack of standardization in fungal biomarker panels. Future directions include AI-driven interpretation of multianalyte data, multiplexed integration of host and pathogen markers, and development of fully cartridge-based systems for near-patient deployment. Collectively, these innovations may shift fungal diagnostics toward earlier, more precise, and patient-tailored interventions, improving outcomes in vulnerable populations.

Proteases are enzymes that catalyse the hydrolysis of peptide bonds in proteins for their functional, modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipeptides off the N-termini of their substrate peptides, typically after proline or alanine. Recently, we showed that human DPP4 and Caenorhabditis elegans DPF-3 have a larger target repertoire in vitro , permitting cleavage after additional amino acids. Here, we use TAILS (Terminal Amine Isotopic Labelling of Substrates) to identify DPF-3 targets in vivo and observe cleavage of MEP-1 after threonine, confirming a broader substrate specificity of DPF-3 also in vivo . Demonstrating physiological relevance, we show that rendering MEP-1 resistant to cleavage disrupts its stability, leading to developmental abnormalities such as defective gonadal migration and reproductive issues. Collectively, our findings highlight a previously unappreciated complexity in the substrate specificity of DPPIV family proteases and suggest that their physiological roles may extend beyond what is currently known. IMPORTANT Manuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. Upon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. The identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. The identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. GUIDELINES For reviewers: https://www.reviewcommons.org/reviewers For authors: https://www.reviewcommons.org/authors CONTACT The Review Commons office can be contacted directly at: office@reviewcommons.org

SUMMARY Transcriptional regulation is a key central mechanism of cell fate determination in developing tissues. The homeobox transcription factor NKX2.2 is an essential regulator of mouse and human pancreatic endocrine development, however its precise molecular role in a human system has not been previously investigated. In this study we generated NKX2.2 null (NKX2.2KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9 technologies and differentiated them towards a pancreatic β cell fate using a stem cell-derived β cell differentiation protocol. Functional and transcriptomic analyses of the hESC-derived pancreatic endocrine cells lacking NKX2.2 revealed similarities and differences compared to the molecular functions of NKX2.2 in mice. In the absence of NKX2.2, the β cell differentiations result in reduced numbers of insulin-producing cells, and the differentiations become skewed towards polyhormonal fates, including cells co-expressing insulin, ghrelin and somatostatin. Deletion of NKX2.2 also eliminates the off-target formation of enterochromaffin cells. Single cell transcriptome analysis of the early endocrine cell population revealed a marked disruption of metabolic pathways that was confirmed by comparative metabolite tracing, providing novel insights into the regulation of early endocrine lineage decisions. Furthermore, NKX2.2 directly regulates several genes in the WNT signaling pathway, suggesting this is a key molecular mechanism through which NKX2.2 regulates these islet cell fate decisions in the human system.

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering ( * PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, * PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2 -mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our * PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Abstract Key Points We present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. * PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. One-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block.

ABSTRACT The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13’s heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first dual-guide RNA system for Cas13 that enhances mismatch discrimination and improves target specificity. This system employs two distinct RNAs—dcrRNA and dtracrRNA—which hybridise to refine target recognition and activation. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, demonstrating its potential for pathogen diagnostics, and successfully discriminated between KRAS G12D and G12C mutations—clinically relevant single-nucleotide variants in cancer diagnosis. These results highlight the dual-guide Cas13 platform’s potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a significant advance over conventional Cas13 systems, offering improved specificity without compromising sensitivity. Its versatility makes it a promising tool for next-generation molecular diagnostics and precision gene editing applications. GRAPHICAL ABSTRACT