STAR Protoc. 2025 Jan 4;6(1):103540. doi: 10.1016/j.xpro.2024.103540. Online ahead of print.

ABSTRACT

Strand-optimized Smart-seq (So-Smart-seq) can capture a comprehensive transcriptome from low-input samples. This technique detects both polyadenylated and non-polyadenylated RNAs, inclusive of repetitive RNAs, while excluding highly abundant ribosomal RNAs. So-Smart-seq preserves strand information and minimizes 5' to 3' coverage bias. We describe steps for the analysis of single mouse preimplantation embryos, including embryo isolation, library preparation, ribosomal cDNA depletion, and initial data processing. The protocol may be adapted for other low-input samples and the detection of small RNAs of <200 nt. For complete details on the use and execution of this protocol, please refer to Wei et al.1.

PMID:39756032 | DOI:10.1016/j.xpro.2024.103540

Nat Commun. 2025 Jan 4;16(1):390. doi: 10.1038/s41467-024-55469-3.

ABSTRACT

Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During the cell cycle, nuclear speckles dissolve following phosphorylation of their protein components. Here, we identify the PP1 family as the phosphatases that counteract kinase-mediated dissolution. PP1 overexpression increases speckle cohesion and leads to retention of mRNA within speckles and the nucleus. Using APEX2 proximity labeling combined with RNA-sequencing, we characterize the recruitment of specific RNAs. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm, particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increases with nuclear speckle cohesion, the ratios of most mRNA species to each other are constant, indicating non-selective retention. We further find that cellular responses to heat shock, oxidative stress, and hypoxia include changes to the phosphorylation and cohesion of nuclear speckles and to mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization.

PMID:39755675 | DOI:10.1038/s41467-024-55469-3

Reprod Biol Endocrinol. 2025 Jan 4;23(1):1. doi: 10.1186/s12958-024-01330-7.

ABSTRACT

BACKGROUND: A didelphic uterus represents a unique and infrequent congenital condition in which a woman possesses two distinct uteri, each with its own cervix. This anomaly arises due to partial or incomplete merging of the Müllerian ducts during the developmental stages in the womb. Accounting for uterine malformations, a didelphic uterus is a relatively rare condition, affecting approximately 0.5-2% of the population and is considered one of the more uncommon types of uterine abnormalities.

METHODS: This case report aims to study the physical separation in uterine didelphys and its impact on endometrial microbiome and inflammation, and the patterns of endometrial receptivity observed.

RESULTS: Endometrial receptivity analyses revealed a similar receptive state in both uteri, both in the early receptive phase. Differential markers of chronic endometritis, including CD138, and MUM1-positive cells, were observed when comparing endometrial biopsies from both uteri. The right uterus exhibited a higher prevalence of these positive cells. Regarding the microbiome, significant differences were found between the uteri, notably in the right uterus, a clear non-dominance of lactobacilli and the presence of genera such as Staphylococcus, Streptococcus, and Acinetobacter. Additionally, the right uterus presented a less 'favourable' microenvironment, a characteristic that was also reflected in the right cervix; both sites presenting less lactobacilli than the left side samples. A distinct metabolomic signature associated with the physical separation of the uteri contributed to the differences in endometrial milieu.

CONCLUSIONS: Our study revealed that physical separation, among other factors in uterus didelphys, affects the endometrial microbiome, metabolome, and inflammatory state, with significant microbiome variation observed between the uteri, although similar endometrial receptivity patterns were noted.

PMID:39755646 | DOI:10.1186/s12958-024-01330-7

Mod Pathol. 2025 Jan 2:100688. doi: 10.1016/j.modpat.2024.100688. Online ahead of print.

ABSTRACT

This manuscript serves as an introduction to a comprehensive seven-part review article series on artificial intelligence (AI) and machine learning (ML) and their current and future influence within pathology and medicine. This introductory review provides a comprehensive grasp of this fast-expanding realm and its potential to transform medical diagnosis, workflow, research, and education. Fundamental terminology employed in AI-ML is covered using an extensive dictionary. The article also provides a broad overview of the main domains in the AI-ML field, encompassing both generative and non-generative (traditional) AI. Thereby serving as a primer to the other six review articles in this series that describe the details about statistics, regulations, bias, ethical dilemmas, and ML-Ops in AI-ML. The intent of these review articles is to better equip individuals who are or will be working in an AI-enabled healthcare system.

PMID:39755237 | DOI:10.1016/j.modpat.2024.100688

Dev Cell. 2025 Jan 2:S1534-5807(24)00758-5. doi: 10.1016/j.devcel.2024.12.019. Online ahead of print.

ABSTRACT

The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell-cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell-cycle-phase markers in the Arabidopsis root. Using single-cell RNA sequencing profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1 phase. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase, followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury, licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.

PMID:39755116 | DOI:10.1016/j.devcel.2024.12.019

Mol Plant. 2025 Jan 2:S1674-2052(25)00003-6. doi: 10.1016/j.molp.2025.01.003. Online ahead of print.

NO ABSTRACT

PMID:39754359 | DOI:10.1016/j.molp.2025.01.003

Cell Commun Signal. 2025 Jan 3;23(1):3. doi: 10.1186/s12964-024-02001-1.

ABSTRACT

BACKGROUND: Renal cell cancer (RCC) is the most common and highly malignant subtype of kidney cancer. Mesenchymal stromal cells (MSCs) are components of tumor microenvironment (TME) that influence RCC progression. The impact of RCC-secreted small non-coding RNAs (sncRNAs) on TME is largely underexplored. Here, we comprehensively analysed the composition of exosomal sncRNAs secreted by RCC cells to identify those that influence MSCs.

METHODS: Exosomal sncRNAs secreted by RCC cells and normal kidney cells were analyzed using RNAseq, followed by qPCR validation. MSCs were treated by conditioned media (CM) derived from RCC cells and transfected with piRNA, followed by the analysis of proliferation, viability, migration and immunocytochemical detection of piRNA. Expression of MSCs genes was evaluated using microarray and qPCR. TCGA data were analyzed to explore the expression of sncRNAs in RCC tumors.

RESULTS: RNAseq revealed 40 miRNAs, 71 tRNAs and four piRNAs that were consistently secreted by RCC cells. qPCR validation using five independent RCC cell lines confirmed that expressions of miR-10b-3p and miR-125a-5p were suppressed, while miR-365b-3p was upregulated in exosomes from RCC cells when compared with normal kidney proximal tubules. The expression of miR-10b-3p and miR-125a-5p was decreased, whereas the expression of miR-365b-3p was increased in RCC tumors and correlated with poor survival of patients. Expressions of tRNA-Glu, tRNA-Gly, and tRNA-Val were the most increased, while tRNA-Gln, tRNA-Leu, and tRNA-Lys were top decreased in RCC exosomes when compared with normal kidney cells. Moreover, hsa_piR_004153, hsa_piR_016735, hsa_piR_019521, and hsa_piR_020365 were consistently upregulated in RCC exosomes. piR_004153 (DQ575660.1; aliases: hsa_piRNA_18299, piR-43772, piR-hsa-5938) was the most highly expressed in exosomes from RCC cells when compared with normal kidney cells. Treatment of MSCs with RCC CM resulted in upregulation of piR_004153 expression. Transfection of MSCs with piR_004153 stimulated their migration and viability, and altered expression of 35 genes, including downregulation of FGF2, SLC7A5, and WISP1. Immunocytochemistry confirmed the nuclear localization of piR_004153 transfected in MSCs.

CONCLUSION: RCC cells secrete multiple sncRNAs, including piR_004153 which targets MSCs, alters expression of FGF2, SLC7A5, and WISP1, and stimulates their motility and viability. To our knowledge, this is the first study showing that cancer-derived piRNA can enhance MSC migration.

PMID:39754169 | DOI:10.1186/s12964-024-02001-1

BMC Genomics. 2025 Jan 3;26(1):4. doi: 10.1186/s12864-024-11191-4.

ABSTRACT

BACKGROUND: Despite the rapid advancement of high-throughput sequencing, simple sequence repeats (SSRs) remain indispensable molecular markers for various applied and research tasks owing to their cost-effectiveness and ease of use. However, existing SSR markers cannot meet the growing demand for research on lotus (Nelumbo Adans.) given their scarcity and weak connections to the lotus genome.

METHODS: Using whole-genome resequencing, active SSR loci were identified throughout the genomes of eight typical Asian lotus. After that, high polymorphism SSR molecular markers were mined from each 2n + 0.5 Mb site on each chromosome (e.g., Chr.1-2.5, 4.5, 6.5 Mb) through four steps: online primer design, primer pair evaluation, agarose gel electrophoresis testing using six Asian lotus, one American lotus, and two their hybrids, and DNA sequence alignment. Finally, the polymerase chain reaction (PCR) efficiency of several SSR markers was validated in 20 Asian temperate lotus, eight Asian tropical lotus, and one American lotus.

RESULTS: A total of 463 SSR markers were developed based on each 2n + 0.5 Mb site of the eight lotus chromosomes (totaling 821.29 Mb). These markers were evenly distributed throughout the lotus genome at a density of 1 SSR per 1.76 Mb. The chromosomal locations of the SSR markers were determined precisely, and the specificity of the primer pairs for each site was verified by sequencing the PCR products. We further provided a set of genome-wide SSR loci, covering 129 per Mb, identified from eight representative Asian lotus, allowing other researchers to independently discover specific SSR markers for particular experiments.

CONCLUSION: These SSR markers, which have a density of 1 SSR marker per 1.76 Mb in this study, will act as a bridge connecting lotus phenotypes with the genome. This work reveals a novel and convenient strategy for developing highly polymorphic SSR markers at any location throughout the lotus genome, and it sheds light on the development of SSR molecular markers in other plant species.

PMID:39754041 | DOI:10.1186/s12864-024-11191-4

BMC Bioinformatics. 2025 Jan 3;26(1):1. doi: 10.1186/s12859-024-06012-0.

ABSTRACT

BACKGROUND: CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications.

RESULTS: We introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. It also provides the capability to design sgRNAs for newly discovered organisms by accepting uploaded design resource. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system.

CONCLUSIONS: We present a web server that allows the construction of genome-scale CRISPRi sgRNA libraries for prokaryotes. It mitigates off-target effects through a robust quality control framework, leveraging prior experimental knowledge within an end-to-end, user-friendly pipeline.

PMID:39754035 | DOI:10.1186/s12859-024-06012-0

Sci Rep. 2025 Jan 2;15(1):495. doi: 10.1038/s41598-024-84900-4.

ABSTRACT

Increasing evidence points toward an essential role for complement activation in the pathogenesis of diabetic kidney disease (DKD). However, the precise molecular mechanisms remain unclear, and the pathway predominantly contributing to complement activation in DKD is of particular interest. In this study, the glomerular proteome, especially the profiles of the complement proteins, was analyzed in kidney biopsies from 40 DKD patients and 10 normal controls using laser microdissection-assisted liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS). The glomerular abundances of three proteins related to classical pathway (CP) (C1q, C1r, C1s), five proteins related to alternative pathway (AP) (CFB, CFH, CFHR1, CFHR3, CFHR5), one common protein related to CP and lectin pathway (LP) (C4), and six proteins related to terminal complement pathway (C3, C5, C6, C7, C8, C9) were significantly increased in DKD. Notably, none of the proteins unique to the lectin complement pathway, including mannose-binding lectin (MBL) and its associated proteins, were detected in DKD glomeruli. Furthermore, the glomerular complement proteins of CP and AP were positively correlated with glomerular pathological grades and proteinuria, and negatively correlated with eGFR in DKD patients. Our results highlight a critical role for complement activation of the CP and AP, rather than the LP, in DKD progression.

PMID:39753879 | DOI:10.1038/s41598-024-84900-4

Sci Rep. 2025 Jan 3;15(1):633. doi: 10.1038/s41598-024-84945-5.

ABSTRACT

Soil is one of the most important reservoirs of antibiotic resistance, global threat that needs to be addressed with the One Health approach. Despite urban parks playing a fundamental role in urban ecosystems, the diffusion, maintenance, and human impact of antibiotic-resistance genes in this substrate are still poorly addressed. To fill in this gap, we adopted a molecular and culturomics approach to study antibiotic resistance in urban parks, accounting for the environmental matrix and the level of urbanization. A higher abundance of efflux-mediated mechanisms in undisturbed environments was observed, while antibiotic alteration or inactivation, and target replacement were more abundant in areas with a higher level of urbanization, also confirmed by significant correlations with anthropogenic features of the environmental matrix. Overall, this study highlights the crucial need to monitor antibiotic resistance in urban parks' soil through a dual molecular and culturomics approach to fully understand and fight antibiotic resistance diffusion.

PMID:39753861 | DOI:10.1038/s41598-024-84945-5

Nat Genet. 2025 Jan 3. doi: 10.1038/s41588-024-02033-w. Online ahead of print.

ABSTRACT

Modern sugarcane, a highly allo-autopolyploid organism, has a very complex genome. In the present study, the karyotype and genome architecture of modern sugarcane were investigated, resulting in a genome assembly of 97 chromosomes (8.84 Gb). The allopolyploid genome was divided into subgenomes from Saccharum officinarum (Soh) and S. spontaneum (Ssh), with Soh dominance in the Saccharum hybrid (S. hybrid). Genome shock affected transcriptome dynamics during allopolyploidization. Analysis of an inbreeding population with 192 individuals revealed the underlying genetic basis of transgressive segregation. Population genomics of 310 Saccharum accessions clarified the breeding history of modern sugarcane. Using the haplotype-resolved S. hybrid genome as a reference, genome-wide association studies identified a potential candidate gene for sugar content from S. spontaneum. These findings illuminate the complex genome evolution of allopolyploids, offering opportunities for genomic enhancements and innovative breeding strategies for sugarcane.

PMID:39753769 | DOI:10.1038/s41588-024-02033-w

Nat Cancer. 2025 Jan 3. doi: 10.1038/s43018-024-00876-0. Online ahead of print.

ABSTRACT

CDKN2A is a tumor suppressor located in chromosome 9p21 and frequently lost in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). How CDKN2A and other 9p21 gene co-deletions affect EAC evolution remains understudied. We explored the effects of 9p21 loss in EACs and cancer progressor and non-progressor BEs with matched genomic, transcriptomic and clinical data. Despite its cancer driver role, CDKN2A loss in BE prevents EAC initiation by counterselecting subsequent TP53 alterations. 9p21 gene co-deletions predict poor patient survival in EAC but not BE through context-dependent effects on cell cycle, oxidative phosphorylation and interferon response. Immune quantifications using bulk transcriptome, RNAscope and high-dimensional tissue imaging showed that IFNE loss reduces immune infiltration in BE, but not EAC. Mechanistically, CDKN2A loss suppresses the maintenance of squamous epithelium, contributing to a more aggressive phenotype. Our study demonstrates context-dependent roles of cancer genes during disease evolution, with consequences for cancer detection and patient management.

PMID:39753721 | DOI:10.1038/s43018-024-00876-0

Trends Genet. 2025 Jan 2:S0168-9525(24)00298-1. doi: 10.1016/j.tig.2024.12.001. Online ahead of print.

ABSTRACT

Genome-wide translational profiling has uncovered the synthesis in human cells of thousands of microproteins, a class of proteins traditionally overlooked in functional studies. Although an increasing number of these microproteins have been found to play critical roles in cellular processes, the functional relevance of the majority remains poorly understood. Studying these low-abundance, often unstable proteins is further complicated by the challenge of disentangling their functions from the noncoding roles of the associated DNA, RNA, and the act of translation. This review highlights recent advances in functional genomics that have led to the discovery of >1000 human microproteins required for optimal cell proliferation. Ongoing technological innovations will continue to clarify the roles and mechanisms of microproteins in both normal physiology and disease, potentially opening new avenues for therapeutic exploration.

PMID:39753408 | DOI:10.1016/j.tig.2024.12.001

J Nucl Med. 2025 Jan 3;66(1):91-97. doi: 10.2967/jnumed.124.268559.

ABSTRACT

Radionuclides used for imaging and therapy can show high molecular specificity in the body with appropriate targeting ligands. We hypothesized that local energy delivered by molecularly targeted radionuclides could chemically activate prodrugs at disease sites while avoiding activation in off-target sites of toxicity. As proof of principle, we tested whether this strategy of radionuclide-induced drug engagement for release (RAiDER) could locally deliver combined radiation and chemotherapy to maximize tumor cytotoxicity while minimizing off-target exposure to activated chemotherapy. Methods: We screened the ability of radionuclides to chemically activate a model radiation-activated prodrug consisting of the microtubule-destabilizing monomethyl auristatin E (MMAE) caged by a radiation-responsive phenyl azide, and we interpreted experimental results using the radiobiology computational simulation suite TOPAS-nBio. RAiDER was evaluated in syngeneic mouse models of cancer using the fibroblast activation protein inhibitor (FAPI) agents [99mTc]Tc-FAPI-34 and [177Lu]Lu-FAPI-04 and the prostate-specific membrane antigen (PSMA) agent [177Lu]Lu-PSMA-617, combined with caged MMAE or caged exatecan. Biodistribution in mice, combined with clinical dosimetry, estimated the relationship between radiopharmaceutical uptake in patients and anticipated concentrations of activated prodrug using RAiDER. Results: RAiDER efficiency varied by 70-fold across radionuclides (99mTc > 111In > 177Lu > 64Cu > 32P > 68Ga > 223Ra > 18F), yielding up to 320 nM prodrug activation/Gy of exposure from 99mTc. Computational simulations implicated low-energy electron-mediated free radical formation as driving prodrug activation. Radionuclide-activated caged MMAE restored the prodrug's ability to destabilize microtubules and increased its cytotoxicity by up to 2,600-fold that of the nonactivated prodrug. Mice treated with [99mTc]Tc-FAPI-34 and caged MMAE accumulated concentrations of activated MMAE that were up to 3,000 times greater in tumors than in other tissues. RAiDER with [99mTc]Tc-FAPI-34 or [177Lu]Lu-FAPI-04 delayed tumor growth, whereas monotherapies did not (P < 0.003). Clinically guided dosimetry suggests sufficient radiation doses can be delivered to activate therapeutically meaningful levels of prodrug. Conclusion: This proof-of-concept study shows that RAiDER is compatible with multiple radionuclides commonly used in nuclear medicine and can potentially improve the efficacy of radiopharmaceutical therapies to treat cancer safely. RAiDER thus shows promise as an effective strategy to treat disseminated malignancies and broadens the capability of radiopharmaceuticals to trigger diverse biologic and therapeutic responses.

PMID:39753366 | DOI:10.2967/jnumed.124.268559

Cancer Cell. 2024 Dec 30:S1535-6108(24)00477-X. doi: 10.1016/j.ccell.2024.12.002. Online ahead of print.

ABSTRACT

Molecular subtypes, such as defined by The Cancer Genome Atlas (TCGA), delineate a cancer's underlying biology, bringing hope to inform a patient's prognosis and treatment plan. However, most approaches used in the discovery of subtypes are not suitable for assigning subtype labels to new cancer specimens from other studies or clinical trials. Here, we address this barrier by applying five different machine learning approaches to multi-omic data from 8,791 TCGA tumor samples comprising 106 subtypes from 26 different cancer cohorts to build models based upon small numbers of features that can classify new samples into previously defined TCGA molecular subtypes-a step toward molecular subtype application in the clinic. We validate select classifiers using external datasets. Predictive performance and classifier-selected features yield insight into the different machine-learning approaches and genomic data platforms. For each cancer and data type we provide containerized versions of the top-performing models as a public resource.

PMID:39753139 | DOI:10.1016/j.ccell.2024.12.002

Structure. 2025 Jan 2;33(1):1-5. doi: 10.1016/j.str.2024.11.012.

ABSTRACT

The data presented at the 9th International Murnau Conference on September 18-21, 2024, the largest recurring structural biology meeting in Central Europe, illustrated the thriving state of the structural biology community. This is largely attributed to the ground-breaking developments over the last decade, which were intensely discussed during the meeting.

PMID:39753099 | DOI:10.1016/j.str.2024.11.012

Food Chem. 2024 Dec 28;470:142638. doi: 10.1016/j.foodchem.2024.142638. Online ahead of print.

ABSTRACT

Fruit preservation materials play an instrumental role in preventing fruit deterioration and extending shelf life. However, existing fresh-keeping materials often prove inadequate in simultaneously achieving antibacterial properties, maintaining freshness, antioxidant effects, good biocompatibility, and prolonged fruit shelf life. Therefore, we present the first preparation of a natural polysaccharide spray hydrogel (Q/O/Zn hydrogel), loaded with chlorogenic acid‑zinc nanoparticles (CA@ZnNPs), utilizing quaternary ammonium insect chitosan (QECS) and oxidized pullulan (OPUL) for the preservation of perishable fruits. The findings demonstrated that the Q/O/Zn hydrogel was highly effective in inhibiting the proliferation of S. aureus and E. coli, while also exhibiting remarkable scavenging capabilities about ABTS and DPPH radicals. Furthermore, the fruit preservation trials demonstrated that the Q/O/Zn hydrogel markedly reduced fruit respiration rates, decay rates, and weight loss across various temperatures while decreasing the species diversity and abundance of harmful bacteria on fruit surfaces. The Q/O/Zn hydrogel is promising for fruit preservation.

PMID:39752739 | DOI:10.1016/j.foodchem.2024.142638

J Proteome Res. 2025 Jan 3. doi: 10.1021/acs.jproteome.4c00775. Online ahead of print.

ABSTRACT

Oxylipins, diverse lipid mediators derived from fatty acids, play key roles in respiratory physiology, but the contribution of lung structural cells to this diverse profile is not well understood. This study aimed to characterize the oxylipin profiles of airway smooth muscle (ASM), lung fibroblasts (HLF), and epithelial (HBE) cells and define how they shift when they are exposed to stimuli related to contractility, fibrosis, and inflammation. Using HPLC-MS/MS, 162 oxylipins were measured in baseline media from cultured human ASM, HLF, and HBE cells as well as after stimulation with modulators of contractility and central regulators of fibrosis/inflammation. At the baseline, ASM and HLF cells had the most similar oxylipin profiles, dominated by oxylipins from cytochrome P450 (CYP450) epoxygenase metabolites. TGFβ stimulation of HLF suppressed CYP450-derived oxylipins, while ASM stimulation increased prostaglandin production. HBE showed the most distinct baseline profile enriched with cyclooxygenase (COX)-derived oxylipins. TGFβ stimulation of HBE increased the level of several oxylipins from CYP450 epoxygenases. These findings highlight the importance of CYP450 oxylipins, which are relatively unexplored in the context of respiratory physiology. By resolving these oxylipin profiles, we enable future respiratory research to understand the function of these oxylipins in regulating physiology, especially in the context of modifying contraction and inflammation.

PMID:39752600 | DOI:10.1021/acs.jproteome.4c00775

Sci Adv. 2025 Jan 3;11(1):eadr7325. doi: 10.1126/sciadv.adr7325. Epub 2025 Jan 3.

ABSTRACT

Lysosomal pH dysregulation is a critical element of the pathophysiology of neurodegenerative diseases, cancers, and lysosomal storage disorders (LSDs). To study the role of lysosomes in pathophysiology, probes to analyze lysosomal size, positioning, and pH are indispensable tools. Here, we developed and characterized a ratiometric genetically encoded lysosomal pH probe, RpH-ILV, targeted to a subpopulation of lysosomal intraluminal vesicles. This subpopulation behaves similarly to the general population of LAMP1-positive vesicles in terms of pH response to pharmacological stresses. In addition, RpH-ILV, which is trafficked to the lysosome via a different cytosolic motif than our previous ratiometric sensor, RpH-LAMP1, is well tolerated by the model organism Drosophila melanogaster, exhibits minimal plasma membrane fluorescence, and reveals sensitivity to the lysosomal damaging agent LLOMe, adding a valuable tool to our repertoire of lysosomal pH sensors.

PMID:39752501 | DOI:10.1126/sciadv.adr7325