Distinct longevity mechanisms across and within species and their association with aging.

Lifespan varies within and across species, but the general principles of its control remain unclear. Here, we conducted multi-tissue RNA-seq analyses across 41 mammalian species, identifying longevity signatures and examining their relationship with transcriptomic biomarkers of aging and established lifespan-extending interventions. An integrative analysis uncovered shared longevity mechanisms within and across species, including downregulated Igf1 and upregulated mitochondrial translation genes, and unique features, such as distinct regulation of the innate immune response and cellular respiration. Signatures of long-lived species were positively correlated with age-related changes and enriched for evolutionarily ancient essential genes, involved in proteolysis and PI3K-Akt signaling. Conversely, lifespan-extending interventions counteracted aging patterns and affected younger, mutable genes enriched for energy metabolism. The identified biomarkers revealed longevity interventions, including KU0063794, which extended mouse lifespan and healthspan. Overall, this study uncovers universal and distinct strategies of lifespan regulation within and across species and provides tools for discovering longevity interventions.

Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis.

Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.

A multimodal atlas of hepatocellular carcinoma reveals convergent evolutionary paths and 'bad apple' effect on clinical trajectory.

BACKGROUND & AIMS: Hepatocellular Carcinoma (HCC) is a highly fatal cancer characterized by high intra-tumor heterogeneity (ITH). A panoramic understanding of its tumor evolution, in relation to its clinical trajectory, may provide novel prognostic and treatment strategies. METHODS: Through the Asia-Pacific Hepatocellular Carcinoma (AHCC) trials group (NCT03267641), we recruited one of the largest prospective cohorts of HCC with over 600 whole genome and transcriptome samples from 123 treatment-naïve patients. RESULTS: Using a multi-region sampling approach, we revealed seven convergent genetic evolutionary paths governed by the early driver mutations, late copy number variations and viral integrations, which stratify patient clinical trajectories after surgical resection. Furthermore, such evolutionary paths shaped the molecular profiles, leading to distinct transcriptomic subtypes. Most significantly, although we found the coexistence of multiple transcriptomic subtypes within certain tumors, patient prognosis was best predicted by the most aggressive cell fraction of the tumor, rather than by overall degree of transcriptomic ITH level - a phenomenon we termed the 'bad apple' effect. Finally, we found that characteristics throughout early and late tumor evolution provide significant and complementary prognostic power in predicting patient survival. CONCLUSIONS: Taken together, our study generated a comprehensive landscape of evolutionary history for HCC and provided a rich multi-omics resource for understanding tumor heterogeneity and clinical trajectories. CLINICAL TRIAL NUMBER: NCT03267641 (Observational cohort) IMPACT AND IMPLICATIONS: This prospective study, utilizing comprehensive multi-sector, multi-omics sequencing and clinical data from surgically resected HCC, reveals critical insights into the role of tumor evolution and intra-tumor heterogeneity (ITH) in determining the prognosis of Hepatocellular Carcinoma (HCC). These findings are invaluable for oncology researchers and clinicians, as they underscore the influence of distinct evolutionary paths and the 'bad apple' effect, where the most aggressive tumor fraction dictates disease progression. These insights not only enhance prognostic accuracy post-surgical resection but also pave the way for developing personalized therapies tailored to specific tumor evolutionary and transcriptomic profiles. The co-existence of multiple sub-types within the same tumor prompts a re-appraisal of the utilities of depending on single samples to represent the entire tumor and suggests the need for clinical molecular imaging. This research thus marks a significant step forward in the clinical understanding and management of HCC, underscoring the importance of integrating tumor evolutionary dynamics and multi-omics biomarkers into therapeutic decision-making.

Achieving ultrahigh fatigue resistance in AlSi10Mg alloy by additive manufacturing.

Since the first discovery of the fatigue phenomenon in the late 1830s, efforts to fight against fatigue failure have continued. Here we report a fatigue resistance phenomenon in nano-TiB2-decorated AlSi10Mg enabled by additive manufacturing. This fatigue resistance mechanism benefits from the three-dimensional dual-phase cellular nanostructure, which acts as a strong volumetric nanocage to prevent localized damage accumulation, thus inhibiting fatigue crack initiation. The intrinsic fatigue strength limit of nano-TiB2-decorated AlSi10Mg was proven to be close to its tensile strength through the in situ fatigue tests of a defect-free microsample. To demonstrate the practical applicability of this mechanism, printed bulk nano-TiB2-decorated AlSi10Mg achieved fatigue resistance more than double those of other additive manufacturing Al alloys and surpassed those of high-strength wrought Al alloys. This strategy of additive-manufacturing-assisted nanostructure engineering can be extended to the development of other dual-phase fatigue-resistant metals.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation.

BACKGROUND & AIMS: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. METHODS: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. RESULTS: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP. CONCLUSIONS: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

Molecular signatures of longevity: Insights from cross-species comparative studies.

Much of the current research on longevity focuses on the aging process within a single species. Several molecular players (e.g. IGF1 and MTOR), pharmacological compounds (e.g. rapamycin and metformin), and dietary approaches (e.g. calorie restriction and methionine restriction) have been shown to be important in regulating and modestly extending lifespan in model organisms. On the other hand, natural lifespan varies much more significantly across species. Within mammals alone, maximum lifespan differs more than 100 fold, but the underlying regulatory mechanisms remain poorly understood. Recent comparative studies are beginning to shed light on the molecular signatures associated with exceptional longevity. These include genome sequencing of microbats, naked mole rat, blind mole rat, bowhead whale and African turquoise killifish, and comparative analyses of gene expression, metabolites, lipids and ions across multiple mammalian species. Together, they point towards several putative strategies for lifespan regulation and cancer resistance, as well as the pathways and metabolites associated with longevity variation. In particular, longevity may be achieved by both lineage-specific adaptations and common mechanisms that apply across the species. Comparing the resulting cross-species molecular signatures with the within-species lifespan extension strategies will improve our understanding of mechanisms of longevity control and provide a starting point for novel and effective interventions.

Phenotypic and molecular features underlying neurodegeneration of motor neurons derived from spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA.

Applications of Acupuncture Therapy in Modulating Plasticity of Central Nervous System.

OBJECTIVE: Acupuncture is widely applied for treatment of various neurological disorders. This manuscript will review the preclinical evidence of acupuncture in mediating neural plasticity, the mechanisms involved. MATERIALS AND METHODS: We searched acupuncture, plasticity, and other potential related words at the following sites: PubMed, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure (CNKI), and VIP information data base. The following keywords were used: acupuncture, electroacupuncture, plasticity, neural plasticity, neuroplasticity, neurogenesis, neuroblast, stem cell, progenitor cell, BrdU, synapse, synapse structure, synaptogenesis, axon, axon regeneration, synaptic plasticity, LTP, LTD, neurotrophin, neurotrophic factor, BDNF, GDNF, VEGF, bFGF, EGF, NT-3, NT-4, NT-5, p75NTR, neurotransmitter, acetylcholine, norepinephrine, noradrenaline, dopamine, monamine. We assessed the effects of acupuncture on plasticity under pathological conditions in this review. RESULTS: Relevant references were reviewed and presented to reflect the effects of acupuncture on neural plasticity. The acquired literatures mainly focused on neurogenesis, alterations of synapses, neurotrophins (NTs), and neurotranimitters. Acupuncture methods mentioned in this article include manual acupuncture and electroacupuncture. CONCLUSIONS: The cumulative evidences demonstrated that acupuncture could induce neural plasticity in rodents exposed to cerebral ischemia. Neural plasticity mediated by acupuncture in other neural disorders, such as Alzheimer's disease, Parkinson's disease, and depression, were also investigated and there is evidence of positive role of acupuncture induced plasticity in these disorders as well. Mediation of neural plasticity by acupuncture is likely associated with its modulation on NTs and neurotransmitters. The exact mechanisms underlying acupuncture's effects on neural plasticity remain to be elucidated. Neural plasticity may be the potential bridge between acupuncture and the treatment of various neurological diseases.

Acupuncture Attenuated Vascular Dementia-Induced Hippocampal Long-Term Potentiation Impairments via Activation of D1/D5 Receptors.

BACKGROUND AND PURPOSE: Emerging evidence suggests that acupuncture could improve cognitive impairment in vascular dementia by enhancing synaptic plasticity in the hippocampus. The purpose of this study is to investigate whether dopamine, a key mediator of synaptic plasticity, is involved in this cognitive improvement. METHODS: Vascular dementia model was established by bilateral common carotid arteries occlusion in male Wistar rats. Three days after the operation, animals received acupuncture treatment for 2 weeks, once daily. The D1/D5 receptors antagonist SCH23390 was administered intraperitoneally 15 minutes before each acupuncture treatment. Morris water maze was examined after acupuncture. Long-term potentiation was studied by an electrophysiological technique. Dopamine and metabolites levels were detected by microdialysis and high-performance liquid chromatography from brain tissue. The expression of D1R and D5R was analyzed by immunofluorescence. RESULTS: Acupuncture remarkably reversed cognitive deficits in 2-vessel occlusion model (2VO) rats, and the acupuncture points Zusanli (ST36) and Baihui (GV20) were confirmed to be the most effective combination. Electrophysiological recording data showed that 2VO-induced impairments of long-term potentiation were prevented by acupuncture. In addition, acupuncture promoted the release of dopamine and its major metabolites in the hippocampus of 2VO rats. The immunofluorescence experiment showed that the decrease of D1R and D5R in hippocampal dentate gyrus region of 2VO rats was reversed by acupuncture. Furthermore, we found that the effects of acupuncture against 2VO-induced impairments in cognition and synaptic plasticity were abolished by SCH23390. CONCLUSIONS: Improvement in cognition and hippocampal synaptic plasticity induced by acupuncture was achieved via activation of D1/D5 receptors in 2VO rats.

Kaposi's Sarcoma-Associated Herpesvirus Inhibitor of cGAS (KicGAS), Encoded by ORF52, Is an Abundant Tegument Protein and Is Required for Production of Infectious Progeny Viruses.

UNLABELLED: Although Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52 (also known as KSHV inhibitor of cGAS [KicGAS]) has been detected in purified virions, the roles of this protein during KSHV replication have not been characterized. Using specific monoclonal antibodies, we revealed that ORF52 displays true late gene expression kinetics and confirmed its cytoplasmic localization in both transfected and KSHV-infected cells. We demonstrated that ORF52 comigrates with other known virion proteins following sucrose gradient centrifugation. We also determined that ORF52 resides inside the viral envelope and remains partially associated with capsid when extracellular virions are treated with various detergents and/or salts. There results indicate that ORF52 is a tegument protein abundantly present in extracellular virions. To characterize the roles of ORF52 in the KSHV life cycle, we engineered a recombinant KSHV ORF52-null mutant virus and found that loss of ORF52 results in reduced virion production and a further defect in infectivity. Upon analysis of the virion composition of ORF52-null viral particles, we observed a decrease in the incorporation of ORF45, as well as other tegument proteins, suggesting that ORF52 is important for the packaging of other virion proteins. In summary, our results indicate that, in addition to its immune evasion function, KSHV ORF52 is required for the optimal production of infectious virions, likely due to its roles in virion assembly as a tegument protein. IMPORTANCE: The tegument proteins of herpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), play key roles in the viral life cycle. Each of the three subfamilies of herpesviruses (alpha, beta, and gamma) encode unique tegument proteins with specialized functions. We recently found that one such gammaherpesvirus-specific protein, ORF52, has an important role in immune evasion during KSHV primary infection, through inhibition of the host cytosolic DNA sensing pathway. In this report, we further characterize ORF52 as a tegument protein with vital roles during KSHV lytic replication. We found that ORF52 is important for the production of infectious viral particles, likely through its role in virus assembly, a critical process for KSHV replication and pathogenesis. More comprehensive investigation of the functions of tegument proteins and their roles in viral replication may reveal novel targets for therapeutic interventions against KSHV-associated diseases.

Mercury Removal by Magnetic Biochar Derived from Simultaneous Activation and Magnetization of Sawdust.

Novel magnetic biochars (MBC) were prepared by one-step pyrolysis of FeCl3-laden biomass and employed for Hg0 removal in simulated combustion flue gas. The sample characterization indicated that highly dispersed Fe3O4 particles could be deposited on the MBC surface. Both enhanced surface area and excellent magnetization property were obtained. With the activation of FeCl3, more oxygen-rich functional groups were formed on the MBC, especially the C═O group. The MBC exhibited far greater Hg0 removal performance compared to the nonmagnetic biochar (NMBC) under N2 + 4% O2 atmosphere in a wide reaction temperature window (120-250 °C). The optimal pyrolysis temperature for the preparation of MBC is 600 °C, and the best FeCl3/biomass impregnation mass ratio is 1.5 g/g. At the optimal temperature (120 °C), the Fe1.5MBC600 was superior in both Hg0 adsorption capacity and adsorption rate to a commercial brominated activated carbon (Br-AC) used for mercury removal in power plants. The mechanism of Hg0 removal was proposed, and there are two types of active adsorption/oxidation sites for Hg0: Fe3O4 and oxygen-rich functional groups. The role of Fe3O4 in Hg0 removal was attributed to the Fe3+(t) coordination and lattice oxygen. The C═O group could act as act as electron acceptors, facilitating the electron transfer for Hg0 oxidation.

Genetic perturbation of mitochondrial function reveals functional role for specific mitonuclear genes, metabolites, and pathways that regulate lifespan.

Altered mitochondrial function is tightly linked to lifespan regulation, but underlying mechanisms remain unclear. Here, we report the chronological and replicative lifespan variation across 167 yeast knock-out strains, each lacking a single nuclear-coded mitochondrial gene, including 144 genes with human homologs, many associated with diseases. We dissected the signatures of observed lifespan differences by analyzing profiles of each strain's proteome, lipidome, and metabolome under fermentative and respiratory culture conditions, which correspond to the metabolic states of replicative and chronologically aging cells, respectively. Examination of the relationships among extended longevity phenotypes, protein, and metabolite levels revealed that although many of these nuclear-encoded mitochondrial genes carry out different functions, their inhibition attenuates a common mechanism that controls cytosolic ribosomal protein abundance, actin dynamics, and proteasome function to regulate lifespan. The principles of lifespan control learned through this work may be applicable to the regulation of lifespan in more complex organisms, since many aspects of mitochondrial function are highly conserved among eukaryotes.

Study on the Relationship between Unexplained Recurrent Abortion and HLA-DQ Gene Polymorphism.

Objective: The study aimed to investigate the relationship between human leukocyte antigen (HLA-DQB1) gene variants and recurrent miscarriage. Methods: HLA-DQ gene polymorphisms (PCR-SSP) were detected in 50 couples with recurrent miscarriage (URSA group) and 30 couples with normal births (control group) using sequence-specific primer-guided polymerase chain reaction. Results: The frequency of the DQB1 ∗ 0303 allele in the URSA group (21.50%) was substantially higher than that of the control group (11.67%) (P=0.0260 0.05, RR = 1.754); however, the frequency of the DQB1 ∗ 0302 allele in the URSA group (4.00%) was substantially lower than that of the control pair (10.00%) (P=0.0318 0.05, RR = 0.400); the frequency of sharing one allele was 46.00% (23/50) in the URSA group and 0.00% (0/30) in the normal control group; the frequency of sharing two alleles was 40.00% (2/50) in the URSA group and 43.33% (13/30) in the normal control group, with no significant difference between the two groups. Conclusion: For the Zhejiang population, HLA-DQB1 ∗ 0303 may be a susceptibility gene for recurrent miscarriage, while HLA-DQB1 ∗ 0302 may be protective against recurrent miscarriage, especially for women.

Moxibustion attenuates neurogenic detrusor overactivity in spinal cord injury rats by inhibiting M2/ATP/P2X3 pathway.

PURPOSE: Activation of muscarinic receptors located in bladder sensory pathways is generally considered to be the primary contributor for driving the pathogenesis of neurogenic detrusor overactivity following spinal cord injury. The present study is undertaken to examine whether moxibustion improves neurogenic detrusor overactivity via modulating the abnormal muscarinic receptor pathway. MATERIALS AND METHODS: Female Sprague-Dawley rats were subjected to spinal cord injury with T9-10 spinal cord transection. Fourteen days later, animals were received moxibustion treatment for one week. Urodynamic parameters and pelvic afferents discharge were measured. Adenosine triphosphate (ATP) content in the voided cystometry fluid was determined. Expressions of M2, M3, and P2X3 receptors in the bladder mucosa were evaluated. RESULTS: Moxibustion treatment prevented the development of detrusor overactivity in spinal cord injury rats, with an increase in the intercontraction interval and micturition pressure threshold and a decrease in afferent activity during filling. The expression of M2 was markedly suppressed by moxibustion, accompanied by a reduction in the levels of ATP and P2X3. M2 receptor antagonist methoctramine hemihydrate had similar effects to moxibustion on bladder function and afferent activity, while the M2-preferential agonist oxotremorine methiodide abolished the beneficial effects of moxibustion. CONCLUSION: Moxibustion is a potential candidate for treating neurogenic bladder overactivity in a rat model of spinal cord injury, possibly through inhibiting the M2/ATP/P2X3 pathway.

Potential Mechanisms and Clinical Effectiveness of Acupuncture in Depression.

Major depressive disorder is the most common mental disorder with significant economic burden and limited treatments. Acupuncture has emerged as a promising non-pharmacological treatment for reducing depressive symptoms. However, the potential mechanisms and clinical effectiveness of acupuncture are not fully understood. This review aimed to: (1) summarize the available evidence on the mechanisms and clinical effectiveness of acupuncture for depression, and then (2) compare with pharmacological interventions, guiding future studies. Studies with animal models of depression and patients have shown that acupuncture could increase hippocampal and network neuroplasticity and decrease brain inflammation, potentially to alleviating depressive disorders. Overall clinical studies indicated that acupuncture could relieve primary depression, particularly milder cases, and was helpful in the management of post-stroke depression, pain-related depression, and postpartum depression both as an isolated and adjunct treatment. It was emphasized that acupuncture combined with antidepressant pharmacological treatment not only enhanced the improvement of primary and secondary depressive symptoms but also reduced the side effects of the medical treatment, which is the main cause for high dropout rates with drug treatment. In summary, substantial evidence from animal and human researches supported the beneficial effect of acupuncture in depression. However, most clinical trials of acupuncture were small, and it is unclear whether their findings can be generalized, so more studies are needed.

Effects of Electroacupuncture at Different Acupoints on Functional Dyspepsia Rats.

Although, acupoint specificity is regarded as the core of scientific issues in electroacupuncture (EA), the difference of EA on treating functional dyspepsia (FD) at different acupoints is unclear. Therefore, this study aims to investigate the different therapeutic effects of EA at lower extremity or abdominal acupoints on the mucosal integrity and lower-inflammatory response in FD. The intragastric administration of iodoacetamide (IA) was performed in 48 rats to establish the FD model. These rats were randomly divided into the control group, the model group and the six EA groups receiving stimulation at the lower extremity (ST36, ST37, and ST39) or abdominal acupoints (ST25, CV4, and CV12) separately. The open-field test (OFT) was measured after 8 weeks of IA, and gastric emptying was evaluated after 10 days of the EA treatment. The local inflammation markers of CD45, eosinophil major basic protein (EMBP), and the tight junction proteins ZO1 and Claudin3 were assessed by immunofluorescence in all groups. Western blot analysis showed that the EMBP and Occludin1 levels in the duodenal. EA at lower extremity acupoint ST36 could improve the gastric emptying. EA at lower extremity acupoints reduced the immunoreactivity of EMBP, but the CD45 was reregulated by the ST37 and ST39 acupoints. The lower extremity acupoints also ameliorated FD-tight junction protein in the expression of Claudin3 and ZO1. However, only the ST36 suppressed the expression of EMBP and recovered the expression of Occludin1. Similarly, the effect of EA at abdominal acupoints was not obvious either in facilitating gastric motility or in improving inflammatory and mucosal injury. EA at lower extremity and abdominal acupoints with the same stimulation parameters had different therapeutic effects in gastric emptying, intestinal mucosal integrity, and inflammation response, thus proving the specificity of acupoints.

Single-cell analysis reveals clonally expanded tumor-associated CD57+ CD8 T cells are enriched in the periphery of patients with metastatic urothelial cancer responding to PD-L1 blockade.

BACKGROUND: A growing body of evidence suggests that T-cell responses against neoantigens are critical regulators of response to immune checkpoint blockade. We previously showed that circulating neoantigen-specific CD8 T cells in patients with lung cancer responding to anti-Programmed death-ligand 1 (PD-L1) (atezolizumab) exhibit a unique phenotype with high expression of CD57, CD244, and KLRG1. Here, we extended our analysis on neoantigen-specific CD8 T cells to patients with metastatic urothelial cancer (mUC) and further profiled total CD8 T cells to identify blood-based predictive biomarkers of response to atezolizumab. METHODS: We identified tumor neoantigens from 20 patients with mUC and profiled their peripheral CD8 T cells using highly multiplexed combinatorial tetramer staining. Another set of patients with mUC treated with atezolizumab (n=30) or chemotherapy (n=40) were selected to profile peripheral CD8 T cells by mass cytometry. Using single-cell transcriptional analysis (single-cell RNA sequencing (scRNA-seq)), together with CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) and paired T-cell receptor (TCR) sequencing, we further characterized peripheral CD8 T cells in a subset of patients (n=16). RESULTS: High frequency of CD57 was observed in neoantigen-specific CD8 T cells in patients with mUC responding to atezolizumab. Extending these findings to bulk CD8 T cells, we found higher frequency of CD57 expressing CD8 T cells before treatment in patients responding to atezolizumab (n=20, p<0.01) but not to chemotherapy. These findings were corroborated in a validation cohort (n=30, p<0.01) and notably were independent of known biomarkers of response. scRNA-seq analysis identified a clonally expanded cluster enriched within CD57+ CD8 T cells in responding patients characterized by higher expression of genes associated with activation, cytotoxicity, and tissue-resident memory markers. Furthermore, compared with CD57- CD8 T cells, TCRs of CD57+ CD8 T cells showed increased overlap with the TCR repertoire of tumor-infiltrating T cells. CONCLUSIONS: Collectively, we show high frequencies of CD57 among neoantigen-specific and bulk CD8 T cells in patients responding to atezolizumab. The TCR repertoire overlap between peripheral CD57+ CD8 T cells and tumor-infiltrating lymphocytes suggest that accumulation of peripheral CD57+ CD8 T cells is reflective of an ongoing antitumor T-cell response. Our findings provide evidence and rationale for using circulating CD8 T cells expressing CD57 as a readily accessible blood-based biomarker for selecting patients with mUC for atezolizumab therapy.

A chromosome-level genome of Antechinus flavipes provides a reference for an Australian marsupial genus with male death after mating.

The 15 species of small carnivorous marsupials that comprise the genus Antechinus exhibit semelparity, a rare life-history strategy in mammals where synchronized death occurs after one breeding season. Antechinus males, but not females, age rapidly (demonstrate organismal senescence) during the breeding season and show promise as new animal models of ageing. Some antechinus species are also threatened or endangered. Here, we report a chromosome-level genome of a male yellow-footed antechinus Antechinus flavipes. The genome assembly has a total length of 3.2 Gb with a contig N50 of 51.8 Mb and a scaffold N50 of 636.7 Mb. We anchored and oriented 99.7% of the assembly on seven pseudochromosomes and found that repetitive DNA sequences occupy 51.8% of the genome. Draft genome assemblies of three related species in the subfamily Phascogalinae, two additional antechinus species (Antechinus argentus and A. arktos) and the iteroparous sister species Murexia melanurus, were also generated. Preliminary demographic analysis supports the hypothesis that climate change during the Pleistocene isolated species in Phascogalinae and shaped their population size. A transcriptomic profile across the A. flavipes breeding season allowed us to identify genes associated with aspects of the male die-off. The chromosome-level A. flavipes genome provides a steppingstone to understanding an enigmatic life-history strategy and a resource to assist the conservation of antechinuses.

Dynamic phenotypic heterogeneity and the evolution of multiple RNA subtypes in hepatocellular carcinoma: the PLANET study.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types.