Creation of memory-memory entanglement in a metropolitan quantum network.

Towards realizing the future quantum internet1,2, a pivotal milestone entails the transition from two-node proof-of-principle experiments conducted in laboratories to comprehensive multi-node set-ups on large scales. Here we report the creation of memory-memory entanglement in a multi-node quantum network over a metropolitan area. We use three independent memory nodes, each of which is equipped with an atomic ensemble quantum memory3 that has telecom conversion, together with a photonic server where detection of a single photon heralds the success of entanglement generation. The memory nodes are maximally separated apart for 12.5 kilometres. We actively stabilize the phase variance owing to fibre links and control lasers. We demonstrate concurrent entanglement generation between any two memory nodes. The memory lifetime is longer than the round-trip communication time. Our work provides a metropolitan-scale testbed for the evaluation and exploration of multi-node quantum network protocols and starts a stage of quantum internet research.

Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and has very few mutations that are shared between different patients. To better understand the intratumoral genetics underlying mutations of ccRCC, we carried out single-cell exome sequencing on a ccRCC tumor and its adjacent kidney tissue. Our data indicate that this tumor was unlikely to have resulted from mutations in VHL and PBRM1. Quantitative population genetic analysis indicates that the tumor did not contain any significant clonal subpopulations and also showed that mutations that had different allele frequencies within the population also had different mutation spectrums. Analyses of these data allowed us to delineate a detailed intratumoral genetic landscape at a single-cell level. Our pilot study demonstrates that ccRCC may be more genetically complex than previously thought and provides information that can lead to new ways to investigate individual tumors, with the aim of developing more effective cellular targeted therapies.

The Phospho-Code Determining Circadian Feedback Loop Closure and Output in Neurospora.

In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology.

Structural and functional reorganization of inhibitory synapses by activity-dependent cleavage of neuroligin-2.

Recent evidence has demonstrated that the transsynaptic nanoscale organization of synaptic proteins plays a crucial role in regulating synaptic strength in excitatory synapses. However, the molecular mechanism underlying this transsynaptic nanostructure in inhibitory synapses still remains unclear and its impact on synapse function in physiological or pathological contexts has not been demonstrated. In this study, we utilized an engineered proteolysis technique to investigate the effects of acute cleavage of neuroligin-2 (NL2) on synaptic transmission. Our results show that the rapid cleavage of NL2 led to impaired synaptic transmission by reducing both neurotransmitter release probability and quantum size. These changes were attributed to the dispersion of RIM1/2 and GABAA receptors and a weakened spatial alignment between them at the subsynaptic scale, as observed through superresolution imaging and model simulations. Importantly, we found that endogenous NL2 undergoes rapid MMP9-dependent cleavage during epileptic activities, which further exacerbates the decrease in inhibitory transmission. Overall, our study demonstrates the significant impact of nanoscale structural reorganization on inhibitory transmission and unveils ongoing modulation of mature GABAergic synapses through active cleavage of NL2 in response to hyperactivity.

East Asian summer monsoon delivers large abundances of very-short-lived organic chlorine substances to the lower stratosphere.

Deep convection in the Asian summer monsoon is a significant transport process for lifting pollutants from the planetary boundary layer to the tropopause level. This process enables efficient injection into the stratosphere of reactive species such as chlorinated very-short-lived substances (Cl-VSLSs) that deplete ozone. Past studies of convective transport associated with the Asian summer monsoon have focused mostly on the south Asian summer monsoon. Airborne observations reported in this work identify the East Asian summer monsoon convection as an effective transport pathway that carried record-breaking levels of ozone-depleting Cl-VSLSs (mean organic chlorine from these VSLSs ~500 ppt) to the base of the stratosphere. These unique observations show total organic chlorine from VSLSs in the lower stratosphere over the Asian monsoon tropopause to be more than twice that previously reported over the tropical tropopause. Considering the recently observed increase in Cl-VSLS emissions and the ongoing strengthening of the East Asian summer monsoon under global warming, our results highlight that a reevaluation of the contribution of Cl-VSLS injection via the Asian monsoon to the total stratospheric chlorine budget is warranted.

Crosstalk between Lys63- and Lys11-polyubiquitin signaling at DNA damage sites is driven by Cezanne.

The establishment of polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.

PELMI: Realize robust DNA image storage under general errors via parity encoding and local mean iteration.

DNA molecules as storage media are characterized by high encoding density and low energy consumption, making DNA storage a highly promising storage method. However, DNA storage has shortcomings, especially when storing multimedia data, wherein image reconstruction fails when address errors occur, resulting in complete data loss. Therefore, we propose a parity encoding and local mean iteration (PELMI) scheme to achieve robust DNA storage of images. The proposed parity encoding scheme satisfies the common biochemical constraints of DNA sequences and the undesired motif content. It addresses varying pixel weights at different positions for binary data, thus optimizing the utilization of Reed-Solomon error correction. Then, through lost and erroneous sequences, data supplementation and local mean iteration are employed to enhance the robustness. The encoding results show that the undesired motif content is reduced by 23%-50% compared with the representative schemes, which improves the sequence stability. PELMI achieves image reconstruction under general errors (insertion, deletion, substitution) and enhances the DNA sequences quality. Especially under 1% error, compared with other advanced encoding schemes, the peak signal-to-noise ratio and the multiscale structure similarity address metric were increased by 10%-13% and 46.8%-122%, respectively, and the mean squared error decreased by 113%-127%. This demonstrates that the reconstructed images had better clarity, fidelity, and similarity in structure, texture, and detail. In summary, PELMI ensures robustness and stability of image storage in DNA and achieves relatively high-quality image reconstruction under general errors.

Partial order relation-based gene ontology embedding improves protein function prediction.

Protein annotation has long been a challenging task in computational biology. Gene Ontology (GO) has become one of the most popular frameworks to describe protein functions and their relationships. Prediction of a protein annotation with proper GO terms demands high-quality GO term representation learning, which aims to learn a low-dimensional dense vector representation with accompanying semantic meaning for each functional label, also known as embedding. However, existing GO term embedding methods, which mainly take into account ancestral co-occurrence information, have yet to capture the full topological information in the GO-directed acyclic graph (DAG). In this study, we propose a novel GO term representation learning method, PO2Vec, to utilize the partial order relationships to improve the GO term representations. Extensive evaluations show that PO2Vec achieves better outcomes than existing embedding methods in a variety of downstream biological tasks. Based on PO2Vec, we further developed a new protein function prediction method PO2GO, which demonstrates superior performance measured in multiple metrics and annotation specificity as well as few-shot prediction capability in the benchmarks. These results suggest that the high-quality representation of GO structure is critical for diverse biological tasks including computational protein annotation.

COSA-1 mediated pro-crossover complex formation promotes meiotic crossing over in C. elegans.

Accurate chromosome segregation during meiosis requires the establishment of at least one crossover (CO) between each pair of homologous chromosomes. CO formation depends on a group of conserved pro-CO proteins, which colocalize at CO-designated sites during late meiotic prophase I. However, it remains unclear whether these pro-CO proteins form a functional complex and how they promote meiotic CO formation in vivo. Here, we show that COSA-1, a key component required for CO formation, interacts with other pro-CO factors, MSH-5 and ZHP-3, via its N-terminal disordered region. Point mutations that impair these interactions do not affect CO designation, but they strongly hinder the accumulation of COSA-1 at CO-designated sites and result in defective CO formation. These defects can be partially bypassed by artificially tethering an interaction-compromised COSA-1 derivate to ZHP-3. Furthermore, we revealed that the accumulation of COSA-1 into distinct foci is required to assemble functional 'recombination nodules'. These prevent early CO-designated recombination intermediates from being dismantled by the RTEL-1 helicase and protect late recombination intermediates, such as Holliday junctions, until they are resolved by CO-specific resolvases. Altogether, our findings provide insight into COSA-1 mediated pro-CO complex assembly and its contribution to CO formation.

Entropic control of the free-energy landscape of an archetypal biomolecular machine.

Biomolecular machines are complex macromolecular assemblies that utilize thermal and chemical energy to perform essential, multistep, cellular processes. Despite possessing different architectures and functions, an essential feature of the mechanisms of action of all such machines is that they require dynamic rearrangements of structural components. Surprisingly, biomolecular machines generally possess only a limited set of such motions, suggesting that these dynamics must be repurposed to drive different mechanistic steps. Although ligands that interact with these machines are known to drive such repurposing, the physical and structural mechanisms through which ligands achieve this remain unknown. Using temperature-dependent, single-molecule measurements analyzed with a time-resolution-enhancing algorithm, here, we dissect the free-energy landscape of an archetypal biomolecular machine, the bacterial ribosome, to reveal how its dynamics are repurposed to drive distinct steps during ribosome-catalyzed protein synthesis. Specifically, we show that the free-energy landscape of the ribosome encompasses a network of allosterically coupled structural elements that coordinates the motions of these elements. Moreover, we reveal that ribosomal ligands which participate in disparate steps of the protein synthesis pathway repurpose this network by differentially modulating the structural flexibility of the ribosomal complex (i.e., the entropic component of the free-energy landscape). We propose that such ligand-dependent entropic control of free-energy landscapes has evolved as a general strategy through which ligands may regulate the functions of all biomolecular machines. Such entropic control is therefore an important driver in the evolution of naturally occurring biomolecular machines and a critical consideration for the design of synthetic molecular machines.

Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

G protein-coupled receptors, including PTHR, are pivotal for controlling metabolic processes ranging from serum phosphate and vitamin D levels to glucose uptake, and cytoplasmic interactors may modulate their signaling, trafficking, and function. We now show that direct interaction with Scribble, a cell polarity-regulating adaptor protein, modulates PTHR activity. Scribble is a crucial regulator for establishing and developing tissue architecture, and its dysregulation is involved in various disease conditions, including tumor expansion and viral infections. Scribble co-localizes with PTHR at basal and lateral surfaces in polarized cells. Using X-ray crystallography, we show that colocalization is mediated by engaging a short sequence motif at the PTHR C-terminus using Scribble PDZ1 and PDZ3 domain, with binding affinities of 31.7 and 13.4 µM, respectively. Since PTHR controls metabolic functions by actions on renal proximal tubules, we engineered mice to selectively knockout Scribble in proximal tubules. The loss of Scribble impacted serum phosphate and vitamin D levels and caused significant plasma phosphate elevation and increased aggregate vitamin D3 levels, whereas blood glucose levels remained unchanged. Collectively these results identify Scribble as a vital regulator of PTHR-mediated signaling and function. Our findings reveal an unexpected link between renal metabolism and cell polarity signaling.

Acetylation of WCC is dispensable for the core circadian clock but differentially regulates acute light responses in Neurospora.

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) have been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is mainly controlled by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

A plasma membrane-localized transporter remobilizes aleurone layer magnesium for seed germination in rice.

The aleurone layer in cereal grains acts as a major reservoir of essential mineral nutrients, significantly influencing seed germination. However, the molecular mechanism underlying the redistribution of nutrients from the aleurone layer in the germinating seed is still not well understood. Here, in rice, we identified a plasma membrane (PM) localized magnesium transporter, MAGNESIUM RELEASE TRANSPORTER 3 (MGR3), is critical for seed germination. OsMGR3 is predominantly expressed in the aleurone layer cells of endosperm, facilitating magnesium remobilization during germination. Non-invasive Micro-test Technology assay data demonstrated that the loss-of-function of OsMGR3 restrained magnesium efflux from the aleurone layer. In the embryo/endosperm grafting experiment, we observed that the mutation of OsMGR3 in the aleurone layer suppressed the growth and differentiation of the embryo during germination. Furthermore, magnesium fluorescence imaging revealed the osmgr3 mutant seeds showed impaired exportation of aleurone layer-stored magnesium to the embryo, consequently delaying germination. Importantly, we discovered that disrupting OsMGR3 could inhibit pre-harvest sprouting without affecting rice yield and quality. Therefore, the magnesium efflux transporter OsMGR3 in the aleurone layer represents a promising genetic target for future agronomic trait improvement.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

Evolutionary trajectory of organelle-derived nuclear DNAs in the Triticum/Aegilops complex species.

Organelle-derived nuclear DNAs, nuclear plastid DNAs (NUPTs), and nuclear mitochondrial DNAs (NUMTs) have been identified in plants. Most, if not all, genes residing in NUPTs/NUMTs (NUPGs/NUMGs) are known to be inactivated and pseudogenized. However, the role of epigenetic control in silencing NUPGs/NUMGs and the dynamic evolution of NUPTs/NUMTs with respect to organismal phylogeny remain barely explored. Based on the available nuclear and organellar genomic resources of wheat (genus Triticum) and goat grass (genus Aegilops) within Triticum/Aegilops complex species, we investigated the evolutionary fates of NUPTs/NUMTs in terms of their epigenetic silencing and their dynamic occurrence rates in the nuclear diploid genomes and allopolyploid subgenomes. NUPTs and NUMTs possessed similar genomic atlas, including (i) predominantly located in intergenic regions and preferential integration to gene regulation regions and (ii) generating sequence variations in the nuclear genome. Unlike nuclear indigenous genes, the alien NUPGs/NUMGs were associated with repressive epigenetic signals, namely high levels of DNA methylation and low levels of active histone modifications. Phylogenomic analyses suggested that the species-specific and gradual accumulation of NUPTs/NUMTs accompanied the speciation processes. Moreover, based on further pan-genomic analyses, we found significant subgenomic asymmetry in the NUPT/NUMT occurrence, which accumulated during allopolyploid wheat evolution. Our findings provide insight into the dynamic evolutionary fates of organelle-derived nuclear DNA in plants.

HDAC1/2/3-mediated downregulation of neurogranin is involved in cognitive impairment in offspring exposed to maternal subclinical hypothyroidism.

Subclinical hypothyroidism (SCH) in pregnancy is the most common form of thyroid dysfunction in pregnancy, which can affect fetal nervous system development and increase the risk of neurodevelopmental disorders after birth. However, the mechanism of the effect of maternal subclinical hypothyroidism on fetal brain development and behavioral phenotypes is still unclear and requires further study. In this study, we constructed a mouse model of maternal subclinical hypothyroidism by exposing dams to drinking water containing 50 ppm propylthiouracil (PTU) during pregnancy and found that its offspring were accompanied by severe cognitive deficits by behavioral testing. Mechanistically, gestational SCH resulted in the upregulation of protein expression and activity of HDAC1/2/3 in the hippocampus of the offspring. ChIP analysis revealed that H3K9ac on the neurogranin (Ng) promoter was reduced in the hippocampus of the offspring of SCH, with a significant reduction in Ng protein, leading to reduced expression levels of synaptic plasticity markers PSD95 (a membrane-associated protein in the postsynaptic density) and SYN (synaptophysin, a specific marker for presynaptic terminals), and impaired synaptic plasticity. In addition, administration of MS-275 (an HDAC1/2/3-specific inhibitor) to SCH offspring alleviated impaired synaptic plasticity and cognitive dysfunction in offspring. Thus, our study suggests that maternal subclinical hypothyroidism may mediate offspring cognitive dysfunction through the HDAC1/2/3-H3K9ac-Ng pathway. Our study contributes to the understanding of the signaling mechanisms underlying maternal subclinical hypothyroidism-mediated cognitive impairment in the offspring.

Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission.

Prolonged myelin deficits contribute to neuron loss and functional impairments after ischaemic stroke.

Ischaemic stroke causes neuron loss and long-term functional deficits. Unfortunately, effective approaches to preserving neurons and promoting functional recovery remain unavailable. Oligodendrocytes, the myelinating cells in the CNS, are susceptible to oxygen and nutrition deprivation and undergo degeneration after ischaemic stroke. Technically, new oligodendrocytes and myelin can be generated by the differentiation of oligodendrocyte precursor cells (OPCs). However, myelin dynamics and their functional significance after ischaemic stroke remain poorly understood. Here, we report numerous denuded axons accompanied by decreased neuron density in sections from ischaemic stroke lesions in human brain, suggesting that neuron loss correlates with myelin deficits in these lesions. To investigate the longitudinal changes in myelin dynamics after stroke, we labelled and traced pre-existing and newly-formed myelin, respectively, using cell-specific genetic approaches. Our results indicated massive oligodendrocyte death and myelin loss 2 weeks after stroke in the transient middle cerebral artery occlusion (tMCAO) mouse model. In contrast, myelin regeneration remained insufficient 4 and 8 weeks post-stroke. Notably, neuronal loss and functional impairments worsened in aged brains, and new myelin generation was diminished. To analyse the causal relationship between remyelination and neuron survival, we manipulated myelinogenesis by conditional deletion of Olig2 (a positive regulator) or muscarinic receptor 1 (M1R, a negative regulator) in OPCs. Deleting Olig2 inhibited remyelination, reducing neuron survival and functional recovery after tMCAO. Conversely, enhancing remyelination by M1R conditional knockout or treatment with the pro-myelination drug clemastine after tMCAO preserved white matter integrity and neuronal survival, accelerating functional recovery. Together, our findings demonstrate that enhancing myelinogenesis is a promising strategy to preserve neurons and promote functional recovery after ischaemic stroke.

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response.