Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

Macrophage IRX3 promotes diet-induced obesity and metabolic inflammation.

Metabolic inflammation is closely linked to obesity, and is implicated in the pathogenesis of metabolic diseases. FTO harbors the strongest genetic association with polygenic obesity, and IRX3 mediates the effects of FTO on body weight. However, in what cells and how IRX3 carries out this control are poorly understood. Here we report that macrophage IRX3 promotes metabolic inflammation to accelerate the development of obesity and type 2 diabetes. Mice with myeloid-specific deletion of Irx3 were protected against diet-induced obesity and metabolic diseases via increasing adaptive thermogenesis. Mechanistically, macrophage IRX3 promoted proinflammatory cytokine transcription and thus repressed adipocyte adrenergic signaling, thereby inhibiting lipolysis and thermogenesis. JNK1/2 phosphorylated IRX3, leading to its dimerization and nuclear translocation for transcription. Further, lipopolysaccharide stimulation stabilized IRX3 by inhibiting its ubiquitination, which amplified the transcriptional capacity of IRX3. Together, our findings identify a new player, macrophage IRX3, in the control of body weight and metabolic inflammation, implicating IRX3 as a therapeutic target.

Modulation of Protein-Interaction States through the Cell Cycle.

Global profiling of protein expression through the cell cycle has revealed subsets of periodically expressed proteins. However, expression levels alone only give a partial view of the biochemical processes determining cellular events. Using a proteome-wide implementation of the cellular thermal shift assay (CETSA) to study specific cell-cycle phases, we uncover changes of interaction states for more than 750 proteins during the cell cycle. Notably, many protein complexes are modulated in specific cell-cycle phases, reflecting their roles in processes such as DNA replication, chromatin remodeling, transcription, translation, and disintegration of the nuclear envelope. Surprisingly, only small differences in the interaction states were seen between the G1 and the G2 phase, suggesting similar hardwiring of biochemical processes in these two phases. The present work reveals novel molecular details of the cell cycle and establishes proteome-wide CETSA as a new strategy to study modulation of protein-interaction states in intact cells.

Single-cell transcriptome analysis of the in vivo response to viral infection in the cave nectar bat Eonycteris spelaea.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8+ effector T cell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in T cell activation and effector function early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R+ population expressing collagen-like genes, which became the predominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies.

OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage.

Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

Single-cell transcriptomic analysis reveals rich pituitary-Immune interactions under systemic inflammation.

The pituitary represents an essential hub in the hypothalamus-pituitary-adrenal (HPA) axis. Pituitary hormone-producing cells (HPCs) release several hormones to regulate fundamental bodily functions under normal and stressful conditions. It is well established that the pituitary endocrine gland modulates the immune system by releasing adrenocorticotropic hormone (ACTH) in response to neuronal activation in the hypothalamus. However, it remains unclear how systemic inflammation regulates the transcriptomic profiles of pituitary HPCs. Here, we performed single-cell RNA-sequencing (scRNA-seq) of the mouse pituitary and revealed that upon inflammation, all major pituitary HPCs respond robustly in a cell type-specific manner, with corticotropes displaying the strongest reaction. Systemic inflammation also led to the production and release of noncanonical bioactive molecules, including Nptx2 by corticotropes, to modulate immune homeostasis. Meanwhile, HPCs up-regulated the gene expression of chemokines that facilitated the communication between the HPCs and immune cells. Together, our study reveals extensive interactions between the pituitary and immune system, suggesting multifaceted roles of the pituitary in mediating the effects of inflammation on many aspects of body physiology.

PHF2 regulates genome topology and DNA replication in neural stem cells via cohesin.

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.

Directed evolution of adeno-associated virus for efficient gene delivery to microglia.

As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.

Atypical behaviour and connectivity in SHANK3-mutant macaques.

Mutation or disruption of the SH3 and ankyrin repeat domains 3 (SHANK3) gene represents a highly penetrant, monogenic risk factor for autism spectrum disorder, and is a cause of Phelan-McDermid syndrome. Recent advances in gene editing have enabled the creation of genetically engineered non-human-primate models, which might better approximate the behavioural and neural phenotypes of autism spectrum disorder than do rodent models, and may lead to more effective treatments. Here we report CRISPR-Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques (Macaca fascicularis) and their F1 offspring. Genotyping of somatic cells as well as brain biopsies confirmed mutations in the SHANK3 gene and reduced levels of SHANK3 protein in these macaques. Analysis of data from functional magnetic resonance imaging revealed altered local and global connectivity patterns that were indicative of circuit abnormalities. The founder mutants exhibited sleep disturbances, motor deficits and increased repetitive behaviours, as well as social and learning impairments. Together, these results parallel some aspects of the dysfunctions in the SHANK3 gene and circuits, as well as the behavioural phenotypes, that characterize autism spectrum disorder and Phelan-McDermid syndrome.

The human pathobiont Malassezia furfur secreted protease Mfsap1 regulates cell dispersal and exacerbates skin inflammation.

Malassezia form the dominant eukaryotic microbial community on the human skin. The Malassezia genus possesses a repertoire of secretory hydrolytic enzymes involved in protein and lipid metabolism which alter the external cutaneous environment. The exact role of most Malassezia secreted enzymes, including those in interaction with the epithelial surface, is not well characterized. In this study, we compared the expression level of secreted proteases, lipases, phospholipases, and sphingomyelinases of Malassezia globosa in healthy subjects and seborrheic dermatitis or atopic dermatitis patients. We observed upregulated gene expression of the previously characterized secretory aspartyl protease MGSAP1 in both diseased groups, in lesional and non-lesional skin sites, as compared to healthy subjects. To explore the functional roles of MGSAP1 in skin disease, we generated a knockout mutant of the homologous protease MFSAP1 in the genetically tractable Malassezia furfur. We observed the loss of MFSAP1 resulted in dramatic changes in the cell adhesion and dispersal in both culture and a human 3D reconstituted epidermis model. In a murine model of Malassezia colonization, we further demonstrated Mfsap1 contributes to inflammation as observed by reduced edema and inflammatory cell infiltration with the knockout mutant versus wildtype. Taken together, we show that this dominant secretory Malassezia aspartyl protease has an important role in enabling a planktonic cellular state that can potentially aid in colonization and additionally as a virulence factor in barrier-compromised skin, further highlighting the importance of considering the contextual relevance when evaluating the functions of secreted microbial enzymes.

Tailoring Interlayer Chiral Exchange by Azimuthal Symmetry Engineering.

Recent theoretical and experimental studies of the interlayer Dzyaloshinskii-Moriya interaction (DMI) have sparked great interest in its implementation into practical magnetic random-access memory (MRAM) devices, due to its capability to mediate long-range chiral spin textures. So far, experimental reports focused on the observation of interlayer DMI, leaving the development of strategies to control interlayer DMI's magnitude unaddressed. Here, we introduce an azimuthal symmetry engineering protocol capable of additive/subtractive tuning of interlayer DMI through the control of wedge deposition of separate layers and demonstrate its capability to mediate field-free spin-orbit torque (SOT) magnetization switching in both orthogonally magnetized and synthetic antiferromagnetically coupled systems. Furthermore, we showcase that the spatial inhomogeneity brought about by wedge deposition can be suppressed by specific azimuthal engineering design, ideal for practical implementation. Our findings provide guidelines for effective manipulations of interlayer DMI strength, beneficial for the future design of SOT-MRAM or other spintronic devices utilizing interlayer DMI.

PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion.

Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

Idiopathic multicentric Castleman disease (iMCD)-idiopathic plasmacytic lymphadenopathy: A distinct subtype of iMCD-not otherwise specified with different clinical features and better survival.

Idiopathic multicentric Castleman disease (iMCD) is subclassified into iMCD-thrombocytopenia, anasarca, reticulin fibrosis, renal dysfunction, organomegaly (TAFRO) and iMCD-not otherwise specified (NOS) according to the Castleman Disease Collaborative Network (CDCN) consensus criteria. With a deeper understanding of iMCD, a group of patients with iMCD-NOS characterised by polyclonal hypergammaglobulinaemia, plasmacytic/mixed-type lymph node histopathology and thrombocytosis has attracted attention. This group of patients has been previously described as having idiopathic plasmacytic lymphadenopathy (IPL). Whether these patients should be excluded from the current classification system lacks sufficient evidence. This retrospective analysis of 228 patients with iMCD-NOS identified 103 (45.2%) patients with iMCD-IPL. The clinical features and outcomes of patients with iMCD-IPL and iMCD-NOS without IPL were compared. Patients with iMCD-IPL showed a significantly higher inflammatory state but longer overall survival. No significant difference in overall survival was observed between severe and non-severe patients in the iMCD-IPL group according to the CDCN severity classification. Compared with lymphoma-like treatments, multiple myeloma-like and IL-6-blocking treatment approaches in the iMCD-IPL group resulted in significantly higher response rates and longer time to the next treatment. These findings highlight the particularities of iMCD-IPL and suggest that it should be considered a new subtype of iMCD-NOS.

Prospective Investigation of Optical Genome Mapping for Prenatal Genetic Diagnosis.

BACKGROUND: Optical genome mapping (OGM) is a novel assay for detecting structural variants (SVs) and has been retrospectively evaluated for its performance. However, its prospective evaluation in prenatal diagnosis remains unreported. This study aimed to prospectively assess the technical concordance of OGM with standard of care (SOC) testing in prenatal diagnosis. METHODS: A prospective cohort of 204 pregnant women was enrolled in this study. Amniotic fluid samples from these women were subjected to OGM and SOC testing, which included chromosomal microarray analysis (CMA) and karyotyping (KT) in parallel. The diagnostic yield of OGM was evaluated, and the technical concordance between OGM and SOC testing was assessed. RESULTS: OGM successfully analyzed 204 cultured amniocyte samples, even with a cell count as low as 0.24 million. In total, 60 reportable SVs were identified through combined OGM and SOC testing, with 22 SVs detected by all 3 techniques. The diagnostic yield for OGM, CMA, and KT was 25% (51/204), 22.06% (45/204), and 18.14% (37/204), respectively. The highest diagnostic yield (29.41%, 60/204) was achieved when OGM and KT were used together. OGM demonstrated a concordance of 95.56% with CMA and 75.68% with KT in this cohort study. CONCLUSIONS: Our findings suggest that OGM can be effectively applied in prenatal diagnosis using cultured amniocytes and exhibits high concordance with SOC testing. The combined use of OGM and KT appears to yield the most promising diagnostic outcomes.

Surgery is a Viable Treatment Option for Stage IE Primary Thyroid Lymphoma.

BACKGROUND: Stage IE primary thyroid lymphoma (PTL) has been diagnosed in approximately half of patients with PTL; however, the optimal treatment for stage IE PTL has not yet been established. METHODS: Stage IE PTL patients were identified from the Surveillance, Epidemiology, and End Results (SEER) database between 1998 and 2019. Thereafter, the disease-specific survival (DSS) and treatment modalities (surgery alone, surgery + radiotherapy (RT) and/or chemotherapy (CT), and RT and/or CT) of these patients were compared by Kaplan-Meier curves and log-rank test after propensity score matching (PSM). Additionally, patients with PTL from the Affiliated Sixth People's Hospital of the Shanghai Jiao Tong University and School of Medicine (Shanghai, China) between 2007 and 2022 were retrospectively analyzed as an external cohort. RESULTS: Among the 1596 patients with PTL from the SEER database, 842 were identified as patients with stage IE PTL, with an average follow-up period of 7.8 years. Pairwise analysis after PSM revealed no significant difference between the DSS of the three treatment groups. A total of 38 patients with PTL were identified in the external cohort, with an average follow-up period of 3.4 years. Compared with the RT and/or CT group, the surgery-alone group showed no significant difference in the incidence of hypothyroidism (p = 0.161) but had significantly fewer treatment-related complications (p = 0.021), shorter treatment duration (p < 0.001), and lower treatment costs (p = 0.025). CONCLUSIONS: The results of our study demonstrate that surgery is a viable treatment option for patients with stage IE PTL.

N6-methyladenosine mRNA methylation positively regulated the response of poplar to salt stress.

As the most abundant form of methylation modification in messenger RNA (mRNA), the distribution of N6-methyladenosine (m6A) has been preliminarily revealed in herbaceous plants under salt stress, but its function and mechanism in woody plants were still unknown. Here, we showed that global m6A levels increased during poplar response to salt stress. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that m6A significantly enriched in the coding sequence region and 3'-untranslated regions in poplar, by recognising the conserved motifs, AGACU, GGACA and UGUAG. A large number of differential m6A transcripts have been identified, and some have been proved involving in salt response and plant growth and development. Further combined analysis of MeRIP-seq and RNA-seq revealed that the m6A hypermethylated and enrich in the CDS region preferred to positively regulate expression abundance. Writer inhibitor, 3-deazaneplanocin A treatment increased the sensitivity of poplar to salt stress by reducing mRNA stability to regulate the expression of salt-responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Furthermore, we verified that the methyltransferase PagFIP37 plays a positively role in the response of poplar to salt stress, overexpressed lines have stronger salt tolerance, while RNAi lines were more sensitive to salt, which relied on regulating mRNA stability in an m6A manner of salt-responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Collectively, these results revealed the regulatory role of m6A methylation in poplar response to salt stress, and revealed the importance and mechanism of m6A methylation in the response of woody plants to salt stress for the first time.

Parametric Analysis of Donor Activation for Glycosylation Reactions.

The chemical synthesis of complex oligosaccharides relies on efficient and highly reproducible glycosylation reactions. The outcome of a glycosylation is contingent upon several environmental factors, such as temperature, acidity, the presence of residual moisture, as well as the steric, electronic, and conformational aspects of the reactants. Each glycosylation proceeds rapidly and with a high yield within a rather narrow temperature range. For better control over glycosylations and to ensure fast and reliable reactions, a systematic analysis of 18 glycosyl donors revealed the effect of reagent concentration, water content, protecting groups, and structure of the glycosyl donors on the activation temperature. With these insights, we parametrize the first step of the glycosylation reaction to be executed reliably and efficiently.

Sirolimus is effective for refractory/relapsed idiopathic multicentric Castleman disease: A single-center, retrospective study.

Refractory/relapsed idiopathic multicentric Castleman disease (R/R iMCD) has limited treatment options. With studies showing increased mTOR activation in iMCD patients, sirolimus becomes an attractive and promising therapy for R/R iMCD. Here we report the results of a retrospective study involving 26 R/R iMCD patients treated with sirolimus-containing regimen. The median age at sirolimus initiation was 40.5 years (23-60), with a median prior treatment line of 2 (1-5). 18 patients (69.2%) achieved symptomatic and biochemical response, with a median time to at least overall partial remission of 1.9 months (0.5-14.6). The median follow-up time from sirolimus initiation was 11.7 months (1.6-50.7) and the median time to next treatment (TTNT) was 46.2 months. No patients died at the end of follow-up. Most of the patients in the cohort are in ongoing responses and continue sirolimus therapy. Sirolimus is well tolerated with minor adverse effects. In conclusion, sirolimus is effective for R/R iMCD patients with good tolerance.

Adsorption Behavior of Ammonia on Strontium Bromide Surface: First-Principles Study.

Thermochemical heat storage based on a gas-solid interaction is an effective long-term energy storage technology and is considered as one of the important technologies for the recovery of industrial waste heat and renewable energy sources such as solar energy. There are many working pairs used for thermochemical heat storage, among which ammonium halides are widely trusted for their good thermodynamic properties. It has attracted a lot of attention in the past decade, but it is still in the laboratory-scale research stage. In this study, the adsorption behavior of strontium bromide surfaces on the atomic scale is investigated using density functional theory with SrBr2/NH3 as the working pair. The optimal adsorption location of ammonia molecules on the strontium bromide surface is determined. Meanwhile, different metal atoms were doped to explore the microscopic factors affecting the adsorption. The energy barrier of the SrBr2/NH3 reaction was 4.507 kcal/mol, which was reduced to 4.145 kcal/mol after doping Mg. The thermodynamics of the Ca atoms doped with SrBr2 were significantly improved, with a reduction in the energy barrier to 0.727 kcal/mol. Comparing the three energy barrier results, Ca doping has a significant optimization effect on the thermal storage process. The results could provide relevant information for the investigation of thermochemical adsorption heat storage, provide insight into the adsorption mechanism of ammonium molecules on strontium bromide, and also facilitate the design of efficient composite adsorbents.