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Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that iPSC human cortical neurons expressing APOE e4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2. via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation, and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.

The rapid and reversible nature of microRNA (miRNA) transcriptional regulation is ideal for implementing global changes to cellular processes and metabolism, a necessary asset for the freeze-tolerant gray tree frog (Dryophytes versicolor). D. versicolor can freeze up to 42% of its total body water during the winter and then thaw completely upon more favorable conditions of spring. Herein, we examined the freeze-specific miRNA responses in the gray tree frog using RBiomirGS, a bioinformatic tool designed for the analysis of miRNA-seq transcriptomics in non-genome sequenced organisms. We identified 11 miRNAs differentially regulated during freezing (miR-140-3p, miR-181a-5p, miR-206-3p, miR-451a, miR-19a-3p, miR-101-3p, miR-30e-5p, miR-142-3p and -5p, miR-21-5p, and miR-34a-5p). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggests these miRNAs play roles in downregulating signaling pathways, apoptosis, and nuclear processes while enhancing ribosomal biogenesis. Overall, these findings point towards miRNA inducing a state of energy conservation by downregulating energy-expensive pathways, while ribosomal biogenesis may lead to prioritization of critical processes for freeze-tolerance survival.

The advent of high-dimensional single-cell data has necessitated the development of dimensionality-reduction tools. t-Distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP) are the two most frequently used approaches, allowing clear visualization of complex single-cell datasets. Despite the need for quantitative comparison, t-SNE and UMAP have largely remained visualization tools due to the lack of robust statistical approaches. Here, we have derived a statistical test for evaluating the difference between dimensionality-reduced datasets using the Kolmogorov-Smirnov test on the distributions of cross entropy of single cells within each dataset. As the approach uses the inter-relationship of single cells for comparison, the resulting statistic is robust and capable of identifying true biological variation. Further, the test provides a valid distance between single-cell datasets, allowing the organization of multiple samples into a dendrogram for quantitative comparison of complex datasets. These results demonstrate the largely untapped potential of dimensionality-reduction tools for biomedical data analysis beyond visualization.

During pre-implantation stages of mammalian development, maternally stored material promotes both the erasure of the sperm and oocyte epigenetic profiles and is responsible for concomitant genome activation. Here, we have utilized single-cell methylome and transcriptome sequencing (scM&T-seq) to quantify both mRNA expression and DNA methylation in oocytes and a developmental series of human embryos at single-cell resolution. We fully characterize embryonic genome activation and maternal transcript degradation and map key epigenetic reprogramming events in developmentally high-quality embryos. By comparing these signatures with early embryos that have undergone spontaneous cleavage-stage arrest, as determined by time-lapse imaging, we identify embryos that fail to appropriately activate their genomes or undergo epigenetic reprogramming. Our results indicate that a failure to successfully accomplish these essential milestones impedes the developmental potential of pre-implantation embryos and is likely to have important implications, similar to aneuploidy, for the success of assisted reproductive cycles.

X chromosome inactivation (XCI) is an essential process, yet it initiates with remarkable diversity in various mammalian species. XIST, the main trigger of XCI, is controlled in the mouse by an interplay of lncRNA genes (LRGs), some of which evolved concomitantly to XIST and have orthologues across all placental mammals. Here, we addressed the functional conservation of human orthologues of two such LRGs, FTX and JPX. By combining analysis of single-cell RNA-seq data from early human embryogenesis with various functional assays in matched human and mouse pluripotent stem- or differentiated post-XCI cells, we demonstrate major functional differences for these orthologues between species, independently of primary sequence conservation. While the function of FTX is not conserved in humans, JPX stands as a major regulator of XIST expression in both species. However, we show that different entities of JPX control the production of XIST at various steps depending on the species. Altogether, our study highlights the functional versatility of LRGs across evolution, and reveals that functional conservation of orthologous LRGs may involve diversified mechanisms of action. These findings represent a striking example of how the evolvability of LRGs can provide adaptative flexibility to constrained gene regulatory networks.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2-STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2-STAT pathway and overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2 mutated MPNs. The non-essential roles of Plek2 under physiologic conditions makes it an ideal target for MPN therapy. Here we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening and cell-based assays followed by the synthesis of analogs. The Plek2 specific small molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2 signaling complex also includes Hsp72 that protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to Plek2 DEP domain. The role of Plek2 in activating the Akt signaling was further confirmed in vivo using a hematopoietic specific Pten knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34 positive cells from MPN patients. Our studies reveal a Plek2-Akt complex that drives cell proliferation and can be targeted by a new class of anti-proliferative compounds for MPN therapy.

Freeze tolerance is an adaptive strategy that wood frogs (Rana sylvatica) use to survive the subzero temperatures of winter. It is characterized by a variety of metabolic and physiological changes that facilitate successful freezing and anoxia. As both mRNA regulation and posttranslation protein modification have been implicated in freeze tolerance, we hypothesized that posttranslational RNA regulation is also involved in coordinating freeze-thaw cycles and metabolic rate depression. As such, we investigated the most abundant RNA modification, adenosine methylation (N -methyladenosine; m A) in wood frog brains during 24 h periods of freezing and anoxia. This was followed by an examination of levels of RNA methyltransferases, demethyltransferases, and the readers of RNA methylation. Despite relative levels of methylation on mRNA remaining constant throughout freezing and anoxia, a significant increase in relative abundance of m A methyltransferases METTL3 and METTL14 was observed. In addition, we investigated the effect of m A RNA methylation on mRNA triaging to stress granules and report a significant increase in stress granule markers TIAR and TIA-1 in both freezing and anoxia. Our findings are the first report of RNA posttranslational regulation during metabolic rate depression in the wood frog brain and suggest that the dynamic RNA methylation observed is not directly linked to mRNA regulation during periods of extreme metabolic reorganization, warranting future investigations.

Scientific research plays a vital role for society, but carries a significant environmental footprint, involving intensive use of energy and resources. Scientists are well placed to understand the unfolding climate and ecological crises, but may not appreciate how heavily their research, and other work-related activities, contribute to emissions and pollution. With the consequences of climate change and ecological breakdown playing out in real time, scientists now have an important, urgent role to play in catalysing solutions. Here, we explore how research organisations can reduce their environmental impact, share useful resources and encourage the global community to engage in making science more sustainable.

DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.

EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models ( cKO, cKO, cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in cKO and cDKO oocytes recapitulated meiotic defects observed in the cKO; however, there was a significant impairment in oocyte maturation and developmental competence in cKO and cDKO oocytes beyond that observed in the cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)-based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in cDKO than cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.

The present work shows a comprehensive chromatographic methods and MS conditions that have been developed according to the chemical properties of each lipid subclass to detect low abundance molecular species. This manuscript shows that the developed methods can detect low and/or very low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipids method, dhCer, dhSPB in the ceramides method, and LPA, LPI, LPG, SPBP in the lysolipids method. Abundant lipid subclasses in human plasma are chromatographically separated from low abundance lipids prior to detection, avoiding the need for derivatisation. Lipid subclasses from the de novo lipogenesis and sphingolipids pathways are presented in this work. Three chromatographic methods here were implemented using a tertiary pumping system to allow for the inclusion of a gradient for analyte separation using A and B pumps, while an isocratic wash elutes interfering compounds. The isocratic wash enabled elution of lipid subclasses not targeted within the method that would otherwise cause background signal in the subsequent sample injection and reduction in column lifetime. Four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches to separate high and low abundance lipid subclasses are described here. An optimised extraction method for lysolipids is also used in addition to Folch extraction in human plasma.

In their attempt to fulfill the wish of having children, women who suffer from fertility issues often undergo assisted reproductive technologies such as ovarian stimulation, which has been associated with adverse health outcomes and imprinting disorders in children. However, given the crucial role of exogenous hormone stimulation in improving human infertility treatments, a more comprehensive analysis of the potential impacts on DNA methylation in embryos following ovarian stimulation is needed. Here, we provide genome-wide DNA methylation profiles of blastocysts generated after superovulation of prepubertal or adult mice, compared with blastocysts derived from non-stimulated adult mice. Additionally, we assessed the impact of the in vitro growth and maturation of oocytes on methylation in blastocysts.

Many bacteria of the genus are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CXC motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CXC were isolated. These isoforms produced increased concentrations of HO in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.

In this editors' corner, the section editors were asked to indicate where they see the autophagy field heading and to suggest what they consider to be key unanswered questions in their specialty area.

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Gene duplication events can drive evolution by providing genetic material for new gene functions, and create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein-coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development.

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Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, -deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

Controlled ovarian stimulation is a necessary step in some assisted reproductive procedures allowing a higher collection of female gametes. However, consequences of this stimulation for the gamete or the offspring have been shown in several mammals. Most studies used comparisons between oocytes from different donors, which may contribute to different responses. In this work, we use the bovine model in which each animal serves as its own control. DNA methylation profiles were obtained by single-cell whole-genome bisulfite sequencing of oocytes from pre-ovulatory unstimulated follicles compared to oocytes from stimulated follicles. Results show that the global percentage of methylation was similar between groups, but the percentage of methylation was lower for non-stimulated oocytes in the imprinted genes , , and and higher in when compared to stimulated oocytes. Differences were also found in CGI of imprinted genes: higher methylation was found among non-stimulated oocytes in (), , , , and . In another region around , the methylation percentage was lower for non-stimulated oocytes when compared to stimulated oocytes. Data drawn from this study might help to understand the molecular reasons for the appearance of certain syndromes in assisted reproductive technologies-derived offspring.

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins T cell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.

Recent studies shed light on new populations and potential roles of Aire and RORγt antigen-presenting cells-including unique subsets with surprising properties-in immune homeostasis and host-microbe interactions.

In preprint