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Yuanyuan Wu,1 Xinmiao Yao,2 Yongliang Jiang,1 Xiaofen He,1 Xiaomei Shao,1 Junying Du,1 Zui Shen,1 Qiaoying He,1 Jianqiao Fang1 1Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, 2The Third Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang, China Abstract: Pain is considered a multidimensional conscious experience that includes a sensory component and a negative affective-motivational component. The negative affective-motivational component of pain is different from the sensory component and amplifies the pain experience. Nowadays, a significant number of preclinical research groups have focused their attention on the affective symptoms of pain. In the present study, we investigated the pain aversion and anxiety-like behavior of the complete Freund’s adjuvant (CFA)-induced chronic pain model. CFA rats experienced spontaneous pain during pain-paired conditioning (pain aversion) and spontaneous pain produces an affective response (anxiety-like behavior). Moreover, pain aversion was gradually attenuated, while the anxiety-like behavior increased in 4 weeks. Therefore, although the negative effect (including pain aversion and anxiety) is always associated with hyperalgesia, the manifestations of negative effect may follow different time courses, which may influence the progress of primary disease. The findings illustrate that targeted therapy should focus on a specific aspect in different stages of pain. Our study emphasizes the necessity of using multiple tests to study pain comorbidities. Keywords: pain, pain aversion, pain affection, anxiety-like behavior, chronic inflammatory pain, rat

Background: Aberrant alternative splicing can generate neoantigens, which can themselves stimulate immune responses and surveillance. Previous methods for quantifying splicing-derived neoantigens are limited by independent references and potential batch effects., Results: Here, we introduce SpliceMutr, a bioinformatics approach and pipeline for identifying splicing derived neoantigens from tumor and normal data. SpliceMutr facilitates the identification of tumor-specific antigenic splice variants, predicts MHC-binding affinity, and estimates splicing antigenicity scores per gene. By applying this tool to genomic data from The Cancer Genome Atlas (TCGA), we generate splicing-derived neoantigens and neoantigenicity scores per sample and across all cancer types and find numerous correlations between splicing antigenicity and well-established biomarkers of anti-tumor immunity. Notably, carriers of mutations within splicing machinery genes have higher splicing antigenicity, which provides support for our approach. Further analysis of splicing antigenicity in cohorts of melanoma patients treated with mono- or combined immune checkpoint inhibition suggests that the abundance of splicing antigens is reduced post-treatment from baseline in patients who progress. We also observe increased splicing antigenicity in responders to immunotherapy, which may relate to an increased capacity to mount an immune response to splicing-derived antigens., Conclusions: We find the splicing antigenicity to be higher in tumor samples when compared to normal, that mutations in the splicing machinery result in increased splicing antigenicity in some cancers, and higher splicing antigenicity is associated with positive response to immune checkpoint inhibitor therapies. Further, this new computational pipeline provides novel analytical capabilities for splicing antigenicity and is openly available for further immuno-oncology analysis.

Maternal use of e-cigarette (e-cig) aerosols poses significant risks to fetal brain development, potentially increasing susceptibility to neurodevelopmental disorders in later life. However, the underlying mechanisms remain incompletely understood. This study aimed to understand the effects of fetal e-cig exposure on DNA methylome and transcriptomic changes in the neonatal brain. Pregnant rats were exposed to e-cig aerosols, and neonatal brains (5 males and 5 females/group) from both control and e-cig-exposed groups were used for experimental analysis. Results indicated that prenatal e-cig exposure altered site-specific DNA methylation patterns at both CpG and CH (non-CpG) sites, predominantly in intergenic and intronic regions, with sex dimorphism in methylation and gene expression changes. Gene ontology analysis revealed that e-cig exposure not only affected neuron projection development and axonogenesis but also altered pathways related to neurodegeneration and long-term depression. These findings provide novel insights into the dynamic changes of CpG and CH methylation induced by e-cig exposure, underscoring the susceptibility of the developing brain to maternal e-cig exposure and its potential implications for developmental disorders and neurodegenerative diseases later in life., (© 2024. The Author(s).)

Primary dysmenorrhea (PDM) refers to the occurrence of spasmodic colicky pain in the lower abdomen without any obvious pelvic pathology, often accompanied by other systemic symptoms, which significantly reduces the quality of life of women with PDM, 45%-95% of menstrual women are affected by it. Despite the high incidence of PDM, it is often not well treated and is ignored by relevant researchers and women themselves. Herbs-partitioned moxibustion (HPM) is a characteristic external therapy in traditional Chinese medicine (TCM), which is widely used to treat diseases in China; it treats diseases by applying a Chinese herbal formula on the patient's acupoints and then igniting moxa sticks on the herbal formula for moxibustion therapy. The primary effectiveness of the herbs utilized in this study is meridian activation and warmth, which encourages qi circulation to relieve pain. Moxibustion's heat stimulation could hasten the infusion of the herbs into the human body. Consequently, HPM, which combines acupoint, herbal, and heat stimulations, is appropriate for treating gynecological disorders, particularly functional diseases such as PDM. This paper provides a detailed description of the procedures and precautions for HPM at the Shenque (CV8) acupoint in female experimental rats, which 0provides an experimental basis for better promoting the application and development of HPM on the navel in PDM.

Background: In utero cigarette smoking/nicotine exposure during pregnancy significantly affects fetal development and increases the risk of cardiovascular disease late in life. However, the underlying molecular mechanisms remain largely unknown. We tested the hypothesis that fetal nicotine aerosol exposure reprograms ischemia-sensitive gene expressions, resulting in increased heart susceptibility to ischemic injury and cardiac dysfunction in adulthood., Methods: Pregnant rats were exposed to chronic intermittent nicotine aerosol (CINA) or saline aerosol control from gestational day 4 to day 21. Experiments were performed on 6-month-old adult offspring., Results: CINA exposure increased ischemia-induced cardiac injury and cardiac dysfunction compared to the control group, which was associated with over- expression of angiotensin II receptor (ATR) protein in the left ventricle (LV) of adult offspring. Meanwhile, CINA exposure up-regulated cardiac TGF-β/SMADs family proteins in the LV. In addition, CINA exposure enhanced cardiac reactive oxygen species (ROS) production and increased the DNA methylation level. The levels of phosphorylated-Akt were upregulated but LC3B-II/I protein abundances were downregulated in the hearts isolated from the CINA-treated group., Conclusion: Fetal nicotine aerosol exposure leads to cardiac dysfunction in response to ischemic stimulation in adulthood. Two molecular pathways are implicated. First, fetal CINA exposure elevates cardiac ATR levels, affecting the TGFβ-SMADs pathway. Second, heightened Angiotensin II/ATR signaling triggers ROS production, leading to DNA hypermethylation, p-Akt activation, and autophagy deficiency. These molecular shifts in cardiomyocytes result in the development of a heart ischemia-sensitive phenotype and subsequent dysfunction in adult offspring., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Cigarette smoking/nicotine exposure in pregnancy shows an increased risk of hypertension in offspring, but the mechanisms are unclear. This study tested the hypothesis that m6A RNA hypomethylation epigenetically regulates vascular NOX (NADPH oxidase) and reactive oxygen species production, contributing to the fetal programming of a hypertensive phenotype in nicotine-exposed offspring., Methods: Pregnant rats were exposed to episodic chronic intermittent nicotine aerosol (CINA) or saline aerosol control from gestational day 4 to day 21, and experiments were performed in 6-month-old adult offspring., Results: Antenatal CINA exposure augmented Ang II (angiotensin II)-stimulated blood pressure response in male, but not female offspring. Moreover, CINA increased vascular NOX2 expression and superoxide production exclusively in male offspring. Inhibition of NOX2 with gp91ds-tat, both ex vivo and in vivo, mitigated the CINA-induced elevation in superoxide production and blood pressure response. Notably, CINA enhanced the expression of vascular m6A demethylase FTO (fat mass and obesity-associated protein), while reducing the total vascular m6A abundance and specific m6A methylation of the NOX2 gene. Additionally, ex vivo inhibition of FTO with FB23-2 attenuated CINA-induced increases in vascular NOX2 expression. In vitro experiments using human umbilical vein endothelial cells demonstrated that nicotine dose-dependently upregulated FTO and NOX2 protein abundance, which were reversed by treatment with the FTO inhibitor FB23-2 or FTO knockdown using siRNAs., Conclusions: This study uncovers a new mechanism: m6A demethylase FTO-mediated epigenetic upregulation of vascular NOX2 signaling in CINA-induced hypertensive phenotype. This insight could lead to a therapeutic target for preventing and treating developmental hypertension programming., Competing Interests: Disclosures None.

Electronic cigarettes (e-cigarettes) are a prevalent alternative to conventional nicotine cigarettes among smokers and people who have never smoked. Increased concentrations of serum free fatty acids (FFAs) are crucial in generating lipotoxicity. We studied the effects of acipimox, an antilipolytic drug, on e-cigarette-induced cardiac dysfunction. C57BL/6J wild-type mice on high fat diet were treated with saline, e-cigarette with 2.4% nicotine [e-cigarette (2.4%)], and e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction were diminished in mice exposed to e-cigarettes (2.4%) compared with saline and acipimox-treated mice. Mice exposed to e-cigarette (2.4%) had increased circulating levels of inflammatory cytokines and FFAs, which were diminished by acipimox. Gene Set Enrichment Analysis revealed that e-cigarette (2.4%)-treated mice had gene expression changes in the G2/M DNA damage checkpoint pathway that was normalized by acipimox. Accordingly, we showed that acipimox suppressed the nuclear localization of phospho-p53 induced by e-cigarette (2.4%). Additionally, e-cigarette (2.4%) increased the apurinic/apyrimidinic sites, a marker of oxidative DNA damage which was normalized by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1 protein levels and 4-hydroxynonenal (4-HNE). These markers of oxidative stress were decreased by acipimox. Therefore, inhibiting lipolysis with acipimox normalizes the physiological changes induced by e-cigarettes and the associated increase in inflammatory cytokines, oxidative stress, and DNA damage., (© 2023. Springer Nature Limited.)

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1β and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: We present here a strategy to identify immunogenic neoantigen candidates from unique amino acid sequences at the junctions of fusion proteins which can serve as targets in the development of tumor vaccines for the treatment of breastcancer., Method: We mined the sequence reads of breast tumor tissue that are usually discarded as discordant paired-end reads and discovered cancer specific fusion transcripts using tissue from cancer free controls as reference. Binding affinity predictions of novel peptide sequences crossing the fusion junction were analyzed by the MHC Class I binding predictor, MHCnuggets. CD8+ T cell responses against the 15 peptides were assessed through in vitro Enzyme Linked Immunospot (ELISpot)., Results: We uncovered 20 novel fusion transcripts from 75 breast tumors of 3 subtypes: TNBC, HER2+, and HR+. Of these, the NSFP1-LRRC37A2 fusion transcript was selected for further study. The 3833 bp chimeric RNA predicted by the consensus fusion junction sequence is consistent with a read-through transcription of the 5'-gene NSFP1-Pseudo gene NSFP1 (NSFtruncation at exon 12/13) followed by trans-splicing to connect withLRRC37A2 located immediately 3' through exon 1/2. A total of 15 different 8-mer neoantigen peptides discovered from the NSFP1 and LRRC37A2 truncations were predicted to bind to a total of 35 unique MHC class I alleles with a binding affinity of IC50<500nM.); 1 of which elicited a robust immune response., Conclusion: Our data provides a framework to identify immunogenic neoantigen candidates from fusion transcripts and suggests a potential vaccine strategy to target the immunogenic neopeptides in patients with tumors carrying the NSFP1-LRRC37A2 fusion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mistretta, Rankothgedera, Castillo, Rao, Holloway, Bhardwaj, El Noafal, Albarracin, El-Zein, Rezaei, Su, Akbani, Shao, Czerniecki, Karchin, Bedrosian and Gunaratne.)

Identifying neoepitopes that elicit an adaptive immune response is a major bottleneck to developing personalized cancer vaccines. Experimental validation of candidate neoepitopes is extremely resource intensive and the vast majority of candidates are non-immunogenic, creating a needle-in-a-haystack problem. Here we address this challenge, presenting computational methods for predicting class I major histocompatibility complex (MHC-I) epitopes and identifying immunogenic neoepitopes with improved precision. The BigMHC method comprises an ensemble of seven pan-allelic deep neural networks trained on peptide-MHC eluted ligand data from mass spectrometry assays and transfer learned on data from assays of antigen-specific immune response. Compared with four state-of-the-art classifiers, BigMHC significantly improves the prediction of epitope presentation on a test set of 45,409 MHC ligands among 900,592 random negatives (area under the receiver operating characteristic = 0.9733; area under the precision-recall curve = 0.8779). After transfer learning on immunogenicity data, BigMHC yields significantly higher precision than seven state-of-the-art models in identifying immunogenic neoepitopes, making BigMHC effective in clinical settings.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1 st to 3 rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2 nd to 3 rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2 nd and 3 rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management., (© 2021. The Author(s).)

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 10 4 cells/3 μL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,β-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca 2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,β-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,β-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Diabetic neuropathic pain (DNP) is a common complication of diabetes. Streptozotocin (STZ)-induced changes of protein in dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) are critical for DNP genesis. However, which proteins change remains elusive. Here, the DNP model was established by a single intraperitoneal injection of STZ, accompanied by increased fasting blood glucose (FBG), decreased body weight (BW), and decreased paw withdrawal latency (PWL). Proteins change in L4-L6 DRGs and SCDH of rats were detected. Western blot and immunofluorescence results showed that expression levels of phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1), Substance P (SP) and calcitonin gene-related peptide (CGRP) in the DRG and the SCDH of rats were increased after STZ injection. A preliminary study from our previous study showed that 2 Hz electroacupuncture (EA) effectively alleviates DNP. However, the analgesic mechanism of EA needs further elucidation. Here, EA at the bilateral Zusanli (ST36) and KunLun (BL60) acupoints was applied for one week, and to investigate the effect on DNP. EA reversed thermal hyperalgesia in DNP rats and downregulated the expression of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH., Competing Interests: The authors declare no conflicts of interest for this work., (Copyright © 2023 Yi-qi Ma et al.)

Tumor mutation burden is an imperfect proxy of tumor foreignness and has therefore failed to consistently demonstrate clinical utility in predicting responses in the context of immunotherapy. We evaluated mutations in regions of the genome that are unlikely to undergo loss in a pan-cancer analysis across 31 tumor types (n = 9,242) and eight immunotherapy-treated cohorts of patients with non-small-cell lung cancer, melanoma, mesothelioma, and head and neck cancer (n = 524). We discovered that mutations in single-copy regions and those present in multiple copies per cell constitute a persistent tumor mutation burden (pTMB) which is linked with therapeutic response to immune checkpoint blockade. Persistent mutations were retained in the context of tumor evolution under selective pressure of immunotherapy and tumors with a high pTMB content were characterized by a more inflamed tumor microenvironment. pTMB imposes an evolutionary bottleneck that cancer cells cannot overcome and may thus drive sustained immunologic tumor control in the context of immunotherapy., (© 2023. The Author(s).)

Stress has been implicated in the etiology of neurological and psychological illnesses. Chronic social isolation (SI) is a psychological stressor that provokes neurobehavioral changes associated with psychiatric disorders, including anxiety disorders. Mitochondria dysfunction and oxidative stress are hallmarks of anxiety pathogenesis. Here we demonstrate the effects of SI-induced stress on mitochondrial function, antioxidative enzymes, autophagy, and brain derivative neurotrophic factor (BDNF). SI induced a reduction in electron transport chain subunits C-I, C-II, and C-VI and an increase in hydrogen peroxide. Treatment with dihydromyricetin (DHM), extracted from Ampelopsis grossedentata , counteracted these changes. A dramatic increase in several primary mitochondrial antioxidative enzymes such as superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1), peroxiredoxin-3 (PRDX3), and glutathione peroxidase 4 (GPX4) was observed after SI and a repeated episode of SI. Both SI and repeated SI induced a reduction in sequestosome 1 (SQSTM1/p62). However, only repeated SI modulated autophagy primary protein beclin-1 (Bcl-1). In addition, SI and repeated SI modulated the BDNF-TrkB signaling pathway and the phosphorylation of the downstream extracellular signal-regulated MAP kinase1/2 (p-Erk p42 and p-Erk p44) cascade. DHM treatment ameliorated these changes. Collectively, we demonstrated that DHM treatment counteracted the effects of SI and repeated SI on antioxidative enzymes, autophagy, and the BDNF-TrkB signaling pathway. These findings highlight the molecular mechanisms that partially explain the anxiolytic effects of DHM., Competing Interests: The authors have nothing to disclose., (© 2022 The Authors.)

Based on the tree-ring increment cores of Pinus tabuliformis collected from the eastern Yinshan Mountains, the tree-ring width chronology was developed. The correlation coefficients were calculated between the chronology and monthly mean temperature and monthly precipitation during the instrumental period of AD 1952-2007. The results showed that the highest correlation was found between the total precipitation from September of previous year to June of current year and the chronology ( r =0.73, n =56, P &lt;0.01). Based on the correlation, the September of previous year to June of current year total precipitation variation was reconstructed in the eastern Yin-shan Mountains during the past 399 years (AD 1619-2017). The reconstruction explained 54.9% of the variation in the total precipitation from September of previous year to June of current year for the calibration period (AD 1952-2007). Both the 'leave-one-out' cross validation and split-period validation showed that the model was relatively robust, with sufficient skills of estimation and high reliability. At the decadal scale, there were four wet periods (AD 1619-1663, AD 1705-1711, AD 1945-1963, and AD 1979-2017)) and four dry periods (AD 1734-1767, AD 1786-1814, AD 1839-1867, and AD 1888-1942) in the past 399 years. Among those periods, the AD 1979-2017 was the wettest period, and AD 1888-1942 was the longest dry duration with the driest period at the late 1920s. Results of power spectral analysis revealed cyclic fluctuations of precipitation series on 2-7 years and 125 years. In addition, the comparison with other reconstructions and spatial correlation analysis indicated that the reconstructed precipitation series well represented regional scale precipitation variation.

A new propaguliferous moss species, Pohliatibetana X.R.Wang & X.M.Shao (Mielichhoferiaceae), from Tibet, southwest China, is described. The new species differs most saliently from other species of Pohlia by its combination of slender plants, loosely attached leaves and axillary solitary, and dark red and flower-like gemmae. In this paper, the line drawings, photographs, habit of the new species are provided and a morphological comparison of it with the similar species is made., (Rui-Hong Wang, A. Jonathan Shaw, Xiao-Ming Shao, Xiao-Rui Wang.)

Introduction: The magnitude of response to immune checkpoint inhibitor (ICI) therapy may be sex-dependent, as females have lower response rates and decreased survival after ICI monotherapy. The mechanisms underlying this sex dimorphism in ICI response are unknown, and may be related to sex-driven differences in the immunogenomic landscape of tumors that shape anti-tumor immune responses in the context of therapy., Methods: To investigate the association of immunogenic mutations with HLA haplotypes, we leveraged whole exome sequence data and HLA genotypes from 482 non-small cell lung cancer (NSCLC) tumors from The Cancer Genome Atlas (TCGA). To explore sex-specific genomic features linked with ICI response, we analyzed whole exome sequence data from patients with NSCLC treated with ICI. Tumor mutational burden (TMB), HLA class I and II restricted immunogenic missense mutation (IMM) load, and mutational smoking signature were defined for each tumor. IMM load was combined with HLA class I and II haplotypes and correlated with therapeutic response and survival following ICI treatment. We examined rates of durable clinical benefit (DCB) for at least six months from ICI treatment initiation. Findings were validated utilizing whole exome sequence data from an independent cohort of ICI treated NSCLC., Results: Analysis of whole exome sequence data from NSCLC tumors of females and males revealed that germline HLA class II diversity (≥9 unique HLA alleles) was associated with higher tumor class II IMM load in females (p=0.01) and not in males (p=0.64). Similarly, in tumors of female patients, somatic HLA class II loss of heterozygosity was associated with increased IMM load (p=0.01) while this association was not observed in tumors in males (p=0.20). In females, TMB (p=0.005), class I IMM load (p=0.005), class II IMM load (p=0.004), and mutational smoking signature (p<0.001) were significantly higher in tumors responding to ICI as compared to non-responding tumors. In contrast, among males, there was no significant association between DCB and any of these features. When IMM was considered in the context of HLA zygosity, high MHC-II restricted IMM load and high HLA class II diversity was significantly associated with overall survival in males (p=0.017)., Conclusions: Inherent sex-driven differences in immune surveillance affect the immunogenomic determinants of response to ICI and likely mediate the dimorphic outcomes with ICI therapy. Deeper understanding of the selective pressures and mechanisms of immune escape in tumors in males and females can inform patient selection strategies and can be utilized to further hone immunotherapy approaches in cancer., Competing Interests: VA receives research funding to the institution from Astra Zeneca and has received research funding to her institution from Bristol-Myers Squibb in the past 5 years. SS has served in a consulting role for Genentech/Roche. KM has received research funding to the institution from Mirati and Bristol Myers Squibb, and she has served in a consulting role for Amgen, Janssen, Mirati, AstraZeneca and Puma. VL has received research funding to the institution from GlaxoSmithKline, Bristol Myers Squibb, Merck, and SeaGen and he has served in a consulting role for Takeda, SeaGen, Bristol Myers Squibb, AstraZeneca, Guardant Health and Takeda. JM has received research funding to the institution from Merck via the Conquer Cancer Young Investigators Award, and has served in a consulting role for MJH Life Sciences, Johnson & Johnson, and Doximity. JF has received research funding to the institution from AstraZeneca, Pfizer, and Bristol Myers Squibb, and has served in a consulting role for Genentech/Roche, Eli Lilly, AstraZeneca, Merck, Takeda, Coherus, Regeneron, and Pfizer. BL has served in a consulting role for AstraZeneca, Daiichi Sankyo, Janssen, Pfizer, Amgen, Takeda, Genentech/Roche, Eli Lilly, Mirati Therapeutics, and Guardant. DE has served in a consulting role for Beyond Spring Pharmaceuticals. CH has received research funding to the institution from AbbVie, Amgen, AstraZeneca, Bristol Myers Squibb, and GlaxoSmithKline, and has served in a consulting role for AbbVie, Amgen, AstraZeneca, Bristol-Myers Squibb, Genentech/Roche, Jannsen and GlaxoSmithKline. JB has received research funding to the institution from AstraZeneca, Bristol Myers Squibb, Genentech/Roche, Merck, RAPT Therapeutics Inc and Revolution Medicines, has served in a consulting role for Amgen, AstraZeneca, BMS, Genentech/Roche, Eli Lilly, GlaxoSmithKline, Merck, Sanofi and Regeneron, and is in the Data and Safety Monitoring Board/Committees of GlaxoSmithKline and Sanofi. PF has received research funding to the institution from AstraZeneca, Bristol Myers Squibb, Novartis, Corvus, Kyowa, and has served as a consultant for Amgen, AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo, Iteos, Janssen, and as a data safety monitoring board member for Polaris and Flame Therapeutics. JN has received research funding from MSD, AstraZeneca, Bristol Myers Squibb, Amgen, Genentech/Roche, Novartis, has served in a consultant/advisory role for Merck, MSD, AstraZeneca, Bristol Myers Squibb, Genentech/Roche, Pfizer, Takeda, Daiichi Sankyo, Kaleido Biosciences, Amgen and Mirati Therapeutics, and is in the Data and Safety Monitoring Board/Committee of Daiichi Sankyo. Under a license agreement between Genentech and the Johns Hopkins University, XS and RK and the University are entitled to royalty distributions related to technology described in the study discussed in this publication. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Scott, Shao, Niknafs, Balan, Pereira, Marrone, Lam, Murray, Feliciano, Levy, Ettinger, Hann, Brahmer, Forde, Karchin, Naidoo and Anagnostou.)

Background: We previously reported a relationship between air pollutants and increased risk of pediatric-onset multiple sclerosis (POMS). Ozone is an air pollutant that may play a role in multiple sclerosis (MS) pathoetiology. CD86 is the only non-HLA gene associated with POMS for which expression on antigen-presenting cells (APCs) is changed in response to ozone exposure., Objectives: To examine the association between county-level ozone and POMS, and the interactions between ozone pollution, CD86 , and HLA - DRB1*15 , the strongest genetic variant associated with POMS., Methods: Cases and controls were enrolled in the Environmental and Genetic Risk Factors for Pediatric MS study of the US Network of Pediatric MS Centers. County-level-modeled ozone data were acquired from the CDC's Environmental Tracking Network. Participants were assigned ozone values based on county of residence. Values were categorized into tertiles based on healthy controls. The association between ozone tertiles and having MS was assessed by logistic regression. Interactions between tertiles of ozone level and the GG genotype of the rs928264 (G/A) single nucleotide polymorphism (SNP) within CD86 , and the presence of DRB1*15:01 ( DRB1*15 ) on odds of POMS were evaluated. Models were adjusted for age, sex, genetic ancestry, and mother's education. Additive interaction was estimated using relative excess risk due to interaction (RERI) and attributable proportions (APs) of disease were calculated., Results: A total of 334 POMS cases and 565 controls contributed to the analyses. County-level ozone was associated with increased odds of POMS (odds ratio 2.47, 95% confidence interval (CI): 1.69-3.59 and 1.95, 95% CI: 1.32-2.88 for the upper two tertiles, respectively, compared with the lowest tertile). There was a significant additive interaction between high ozone tertiles and presence of DRB1*15 , with a RERI of 2.21 (95% CI: 0.83-3.59) and an AP of 0.56 (95% CI: 0.33-0.79). Additive interaction between high ozone tertiles and the CD86 GG genotype was present, with a RERI of 1.60 (95% CI: 0.14-3.06) and an AP of 0.37 (95% CI: 0.001-0.75) compared to the lowest ozone tertile. AP results indicated that approximately half of the POMS risk in subjects can be attributed to the possible interaction between higher county-level ozone carrying either DRB1*15 or the CD86 GG genotype., Conclusions: In addition to the association between high county-level ozone and POMS, we report evidence for additive interactions between higher county-level ozone and DRB1*15 and the CD86 GG genotype. Identifying gene-environment interactions may provide mechanistic insight of biological processes at play in MS susceptibility. Our work suggests a possible role of APCs for county-level ozone-induced POMS risk.

Background: Whereas human leukocyte antigen (HLA) class I mutation-associated neoantigen burden has been linked with response to immune checkpoint blockade (ICB), the role of HLA class II-restricted neoantigens in clinical responses to ICB is less studied. We used computational approaches to assess HLA class II immunogenic mutation (IMM) burden in patients with melanoma and lung cancer treated with ICB., Patients and Methods: We analyzed whole-exome sequence data from four cohorts of ICB-treated patients with melanoma (n = 110) and non-small-cell lung cancer (NSCLC) (n = 123). MHCnuggets, a neural network-based model, was applied to estimate HLA class II IMM burdens and cellular fractions of IMMs were calculated to assess mutation clonality. We evaluated the combined impact of HLA class II germline genetic variation and class II IMM burden on clinical outcomes. Correlations between HLA class II IMM burden and density of tumor-infiltrating lymphocytes were computed from expression data., Results: Responding tumors harbored a significantly higher HLA class II IMM burden for both melanoma and NSCLC (P ≤ 9.6e-3). HLA class II IMM burden was correlated with longer survival, particularly in the NSCLC cohort and in the context of low intratumoral IMM heterogeneity (P < 0.001). HLA class I and II IMM landscapes were largely distinct suggesting a complementary role for class II IMMs in tumor rejection. A higher HLA class II IMM burden was associated with CD4+ T-cell infiltration and programmed death-ligand 1 expression. Transcriptomic analyses revealed an inflamed tumor microenvironment for tumors harboring a high HLA class II IMM burden., Conclusions: HLA class II IMM burden identified patients with NSCLC and melanoma that attained longer survival after ICB treatment. Our findings suggest that HLA class II IMMs may impact responses to ICB in a manner that is distinct and complementary to HLA class I-mediated responses., Competing Interests: Disclosure Under a license agreement between Genentech and the Johns Hopkins University, XMS and RK and the University are entitled to royalty distributions related to technology described in the study discussed in this publication. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. VA receives research funding to her institution from AstraZeneca and has received research funding to her institution from Bristol Myers Squibb in the past 5 years. VEV is a founder of Delfi Diagnostics and Personal Genome Diagnostics, serves on the Board of Directors and as a consultant for both organizations, and owns Delfi Diagnostics and Personal Genome Diagnostics stock, which are subject to certain restrictions under university policy. Additionally, Johns Hopkins University owns equity in Delfi Diagnostics and Personal Genome Diagnostics. VEV is an advisor to Bristol Myers Squibb, Genentech, Merck, and Takeda Pharmaceuticals. Within the last 5 years, VEV has been an advisor to Daiichi Sankyo, Janssen Diagnostics, and Ignyta (these arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies). JRW is founder and owner of Resphera Biosciences LLC, and serves as a consultant to Personal Genome Diagnostics Inc. All other authors have declared no conflicts of interest. Ethical approval Regulatory and ethical considerations are described in detail in the original publications from which published data were retrieved to perform the analyses described in this study., (Copyright © 2022 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Social isolation induces stress, anxiety, and mild cognitive impairment that could progress towards irreversible brain damage. A probable player in the mechanism of social isolation-induced anxiety is astrocytes, specialized glial cells that support proper brain function. Using a social isolation mouse model, we observed worsened cognitive and memory abilities with reductions of Object Recognition Index (ORI) in novel object recognition test and Recognition Index (RI) in novel context recognition test. Social isolation also increased astrocyte density, reduced astrocyte size with shorter branches, and reduced morphological complexity in the hippocampus. Dihydromyricetin, a flavonoid that we previously demonstrated to have anxiolytic properties, improved memory/cognition and restored astrocyte plasticity in these mice. Our study indicates astrocytic involvement in social isolation-induced cognitive impairment as well as anxiety and suggest dihydromyricetin as an early-stage intervention against anxiety, cognitive impairment, and potential permanent brain damage., (© 2022. The Author(s).)

Electronic cigarettes or e-cigarettes are the most frequently used tobacco product among adolescents. Despite the widespread use of e-cigarettes and the known detrimental cardiac consequences of nicotine, the effects of e-cigarettes on the cardiovascular system are not well-known. Several in vitro and in vivo studies delineating the mechanisms of the impact of e-cigarettes on the cardiovascular system have been published. These include mechanisms associated with nicotine or other components of the aerosol or thermal degradation products of e-cigarettes. The increased hyperlipidemia, sympathetic dominance, endothelial dysfunction, DNA damage, and macrophage activation are prominent effects of e-cigarettes. Additionally, oxidative stress and inflammation are unifying mechanisms at many levels of the cardiovascular impairment induced by e-cigarette exposure. This review outlines the contribution of e-cigarettes in the development of cardiovascular diseases and their molecular underpinnings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Espinoza-Derout, Shao, Lao, Hasan, Rivera, Jordan, Echeverria, Roos, Sinha-Hikim and Friedman.)

Background: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country's mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system., Methods: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured., Results: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes., Conclusion: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety., (© 2022. The Author(s).)

Opiates produce a strong rewarding effect, but abstinence from opiate use emerges with severe negative emotions. Depression is one of the most frequent emotion disorders associated with opiate abstinence, which is thought to be a main cause for relapse. However, neurobiological bases of such an aversive emotion processing are poorly understood. Here, we find that morphine abstinence activates κ-opioid receptors (KORs) by increasing endogenous KOR ligand dynorphin expression in the amygdala, which in turn facilitates glutamate transporter 1 (GLT1) expression by activation of p38 mitogen-activated protein kinase (MAPK). Upregulation of GLT1 expression contributes to opiate-abstinence-elicited depressive-like behaviors through modulating amygdalar glutamatergic inputs to the nucleus accumbens (NAc). Intra-amygdala injection of GLT1 inhibitor DHK or knockdown of GLT1 expression in the amygdala significantly suppresses morphine-abstinence-induced depressive-like behaviors. Pharmacological and pharmacogenetic activation of amygdala-NAc projections prevents morphine-abstinence-induced behaviors. Overall, our study provides key molecular and circuit insights into the mechanisms of depression associated with opiate abstinence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Maternal e-cigarette (e-cig) exposure is a pressing perinatal health concern. Emerging evidence reveals its potential adverse impacts on brain development in offspring, yet the underlying mechanisms are poorly understood. The present study tested the hypothesis that fetal e-cig exposure induces an aberrant DNA methylation profile in the developing brain, leading to alteration of autophagic flux signaling and programming of a sensitive phenotype to neonatal hypoxic-ischemic encephalopathy (HIE). Pregnant rats were exposed to chronic intermittent e-cig aerosol. Neonates were examined at the age of 9 days old. Maternal e-cig exposure decreased the body weight and brain weight but enhanced the brain-to-body weight ratio in the neonates. E-cig exposure induced a gender-dependent increase in hypoxic-ischemia-induced brain injury in male neonates associated with enhanced reactive oxygen species (ROS) activity. It differentially altered DNA methyltransferase expression and enhanced both global DNA methylation levels and specific CpG methylation at the autophagy-related gene 5 (ATG5) promoter. In addition, maternal e-cig exposure caused downregulations of ATG5, microtubule-associated protein 1 light chain 3β, and sirtuin 1 expression in neonatal brains. Of importance, knockdown of ATG5 in neonatal pups exaggerated neonatal HIE. In conclusion, the present study reveals that maternal e-cig exposure downregulates autophagy-related gene expression via DNA hypermethylation, leading to programming of a hypoxic-ischemic sensitive phenotype in the neonatal brain.

Mesothelioma is a rare and fatal cancer with limited therapeutic options until the recent approval of combination immune checkpoint blockade. Here we report the results of the phase 2 PrE0505 trial ( NCT02899195 ) of the anti-PD-L1 antibody durvalumab plus platinum-pemetrexed chemotherapy for 55 patients with previously untreated, unresectable pleural mesothelioma. The primary endpoint was overall survival compared to historical control with cisplatin and pemetrexed chemotherapy; secondary and exploratory endpoints included safety, progression-free survival and biomarkers of response. The combination of durvalumab with chemotherapy met the pre-specified primary endpoint, reaching a median survival of 20.4 months versus 12.1 months with historical control. Treatment-emergent adverse events were consistent with known side effects of chemotherapy, and all adverse events due to immunotherapy were grade 2 or lower. Integrated genomic and immune cell repertoire analyses revealed that a higher immunogenic mutation burden coupled with a more diverse T cell repertoire was linked to favorable clinical outcome. Structural genome-wide analyses showed a higher degree of genomic instability in responding tumors of epithelioid histology. Patients with germline alterations in cancer predisposing genes, especially those involved in DNA repair, were more likely to achieve long-term survival. Our findings indicate that concurrent durvalumab with platinum-based chemotherapy has promising clinical activity and that responses are driven by the complex genomic background of malignant pleural mesothelioma., (© 2021. The Author(s).)

Evaluating the impacts of human activity on river runoff has important implications for regional water resource management. Here, we used seven tree-ring width chronologies to establish a regional mean tree-ring width chronology from the northern mountain of Delingha, Qaidam Basin. We conducted the correlation, moving correlation and regression analysis of regional mean tree-ring width chronology with runoff data from Bayin River gauge station. Then, we stimulated the June runoff of Bayin River from 1956 to 2002. The results showed that the highest correlation coefficient was found for June runoff ( r =0.63, P <0.01), and their moving correlation coefficient decreased after 1986. Based on the stable relationship between tree-ring width chronology and the June runoff during 1956-1986, we built the reconstruction function, which was explained 50.8% of observed runoff. The stimulated runoff during 1987 to 2002 was significantly higher than the observed runoff (3.01 m 3 ·s -1 , P <0.001). These results indicated that human activity from the upper river had significant impacts on Bayin River runoff. Human activity should be considered as an important factor to protect security of sustainable water resource utilization for future water resource development and utilization in Bayin River region.

Chronic pain easily leads to concomitant mood disorders, and the excitability of anterior cingulate cortex (ACC) pyramidal neurons (PNs) is involved in chronic pain-related anxiety. However, the mechanism by which PNs regulate pain-related anxiety is still unknown. The GABAergic system plays an important role in modulating neuronal activity. In this paper, we aimed to study how the GABAergic system participates in regulating the excitability of ACC PNs, consequently affecting chronic inflammatory pain-related anxiety. A rat model of CFA-induced chronic inflammatory pain displayed anxiety-like behaviors, increased the excitability of ACC PNs, and reduced inhibitory presynaptic transmission; however, the number of GAD65/67 was not altered. Interestingly, intra-ACC injection of the GABA A R agonist muscimol relieved anxiety-like behaviors but had no effect on chronic inflammatory pain. Intra-ACC injection of the GABA A R antagonist picrotoxin induced anxiety-like behaviors but had no effect on pain in normal rats. Notably, chemogenetic activation of GABAergic neurons in the ACC alleviated chronic inflammatory pain and pain-induced anxiety-like behaviors, enhanced inhibitory presynaptic transmission, and reduced the excitability of ACC PNs. Chemogenetic inhibition of GABAergic neurons in the ACC led to pain-induced anxiety-like behaviors, reduced inhibitory presynaptic transmission, and enhanced the excitability of ACC PNs but had no effect on pain in normal rats. We demonstrate that the GABAergic system mediates a reduction in inhibitory presynaptic transmission in the ACC, which leads to enhanced excitability of pyramidal neurons in the ACC and is associated with chronic inflammatory pain-related anxiety., (© 2021. The Author(s).)

A SMBBR was established to treat medium ammonium under room temperature. Results showed that TN load can reach 0.16 kg·(m 3 ·d) -1 , and the average TN removal efficiency was (51.58±6.80)% in the SMBBR with an influent ammonia concentration of 100 mg·L -1 and DO of 0.4-0.7 mg·L -1 . AOB, ANAMMOX, and NOB activity reached (2253.21±502.10) mg·(m 2 ·d) -1 , (4847.46±332.89) mg·(m 2 ·d) -1 , and (1455.17±473.83) mg·(m 2 ·d) -1 , and ANAMMOX and AOB bacteria were found to develop a good collaborative relationship. Quantitative PCR results showed that the relative abundance of ANAMMOX, AOB and NOB were 11.57%, 1.01% and 0.94%, respectively. The stable operation of single stage partial nitritation-ANAMMOX process provide an alternative technology for medium ammonia wastewater.

Ethnopharmacological Relevance: Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABA A receptors (GABA A Rs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABA A Rs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABA A R complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation., Materials and Methods: The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABA A Rs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABA A Rs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids., Results: Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABA A Rs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, K i  = 0.59 μM), a flavone that binds to recombinant α1-3/5 subunit-containing GABA A Rs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABA A Rs., Conclusions: This present study indicated that the flavones, especially DMS, are novel GABA A R ligands and therapeutic potential candidates for anxiety., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Epidemiological studies have shown an increased risk of cardiovascular diseases in children born to mothers who smoked during pregnancy. The cardiovascular risk in the offspring associated with in utero nicotine exposure is further exaggerated by maternal obesity. The consumption of electronic cigarettes (e-cigarettes) is alarmingly increasing among adolescents and young adults without the knowledge of their harmful health effects. There has also been a substantial increase in e-cigarette use by women of reproductive age. This study investigates the detrimental effects of gestational exposure of e-cigarette and a high-fat diet (HFD) on neonatal hearts. Time-mated pregnant mice were fed a HFD and exposed to saline or e-cigarette aerosol with 2.4% nicotine from embryonic day 4 (E4) to E20. We demonstrated that in utero exposure of e-cigarettes and HFD from E4 to E20 triggers cardiomyocyte (CM) apoptosis in the offspring at postnatal day1 (PND1), PND3, and PND14. Induction of CM apoptosis following gestational exposure of e-cigarettes and HFD was associated with inactivation of AMP-activated protein kinase (AMPK), increased cardiac oxidative stress coupled with perturbation of cardiac BAX/BCL-2 ratio and activation of caspase 3 at PND 14. Electron microscopy further revealed that left ventricles of pups at PND14 after e-cigarette exposure exhibited apoptotic nuclei, convoluted nuclear membranes, myofibrillar derangement, and enlarged mitochondria occasionally showing signs of crystolysis, indicative of cardiomyopathy and cardiac dysfunction. Our results show profound adverse effects of prenatal exposure of e-cigarette plus HFD in neonatal hearts that may lead to long-term adverse cardiac consequences in the adult., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we incorporate analyses of genome-wide sequence and structural alterations with pre- and on-therapy transcriptomic and T cell repertoire features in immunotherapy-naive melanoma patients treated with immune checkpoint blockade. Although tumor mutation burden is associated with improved treatment response, the mutation frequency in expressed genes is superior in predicting outcome. Increased T cell density in baseline tumors and dynamic changes in regression or expansion of the T cell repertoire during therapy distinguish responders from non-responders. Transcriptome analyses reveal an increased abundance of B cell subsets in tumors from responders and patterns of molecular response related to expressed mutation elimination or retention that reflect clinical outcome. High-dimensional genomic, transcriptomic, and immune repertoire data were integrated into a multi-modal predictor of response. These findings identify genomic and transcriptomic characteristics of tumors and immune cells that predict response to immune checkpoint blockade and highlight the importance of pre-existing T and B cell immunity in therapeutic outcomes., Competing Interests: V.A. and J.T. receive research funding from Bristol-Myers Squibb. J.T. serves as a consultant/advisory board member to Bristol-Myers Squibb, Merck, Astra Zeneca, and Compugen. J.R.W. is a consultant for Personal Genome Diagnostics; is the founder and owner of Resphera Biosciences; and holds patents, royalties, or other intellectual property from Personal Genomic Diagnostics. A.B. receives honoraria from Proscia and Corista; is a consultant of Bristol-Myers Squibb, Genentech, and Bayer; and receives research funding from Genentech. C.G. has patents, royalties, or other intellectual property from Karyopharm and Arcus. A.R. has received honoraria from consulting with Amgen, Bristol-Myers Squibb, Chugai, Genentech, Merck, Novartis, Roche, and Sanofi; is or has been a member of the scientific advisory board and holds stock in Advaxis, Arcus Biosciences, Bioncotech Therapeutics, Compugen, CytomX, Five Prime, FLX-Bio, ImaginAb, Isoplexis, Kite-Gilead, Lutris Pharma, Merus, PACT Pharma, Rgenix, and Tango Therapeutics; and has received research funding from Agilent and from Bristol-Myers Squibb through Stand Up to Cancer (SU2C). P.R.-M. and M.W.-R. are employees of Bristol-Myers Squibb. D.M.P. and S.L.T. report stock and other ownership interests in Aduro Biotech, DNAtrix, Dracen Pharmaceuticals, Dragonfly Therapeutics, Ervaxx, Five Prime Therapeutics, Potenza Therapeutics, RAPT, Tizona Therapeutics, Trieza Therapeutics, and WindMIL; a consulting or advisory role in Amgen, DNAtrix, Dragonfly Therapeutics, Dynavax, Ervaxx, Five Prime Therapeutics, Immunocore, Immunomic Therapeutics, Janssen Pharmaceuticals, MedImmune/AstraZeneca, Merck, RAPT, and WindMIL; research grants from Bristol-Myers Squibb and Compugen; patents, royalties, and/or other intellectual property through their institution with Aduro Biotech, Arbor Pharmaceuticals, Bristol-Myers Squibb, Immunomic Therapeutics, NexImmune, and WindMIL; and travel, accommodations, and expenses from Bristol-Myers Squibb and Five Prime Therapeutics. V.E.V. is a founder of Delfi Diagnostics and Personal Genome Diagnostics, serves on the Board of Directors and as a consultant for both organizations, and owns Delfi Diagnostics and Personal Genome Diagnostics stock, which are subject to certain restrictions under university policy. Additionally, Johns Hopkins University owns equity in Delfi Diagnostics and Personal Genome Diagnostics. V.E.V. is an advisor to Bristol-Myers Squibb, Genentech, Merck, and Takeda Pharmaceuticals. Within the last 5 years, V.E.V. has been an advisor to Daiichi Sankyo, Janssen Diagnostics, and Ignyta. These arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies., (© 2020 The Authors.)

In spite of the widespread use of electronic cigarettes, also known as e-cigarettes, and the proposed adverse cardiac effects of nicotine, the detrimental effects of e-cigarettes on the heart are not well known. This study examines the detrimental effects of e-cigarettes with nicotine at doses that yield circulating nicotine and cotinine in the ranges similar to the levels found in habitual smokers, and a high fat diet (HFD) on cardiac structure and function in a commonly used model of diet-induced obesity (DIO). C57BL/6J mice on an HFD were exposed to e-cigarette in the presence (2.4% nicotine) or absence (0% nicotine) of nicotine and saline aerosol for 12 weeks. Echocardiographic data demonstrated a decrease in left ventricular (LV) fractional shortening, LV ejection fraction, and velocity of circumferential fiber shortening (VCF) in mice treated with e-cigarette (2.4% nicotine) compared to e-cigarette (0% nicotine) or saline exposed mice. Cardiomyocytes (CMs) of mice treated with e-cigarette (2.4% nicotine) exhibited LV abnormalities, including lipid accumulation (ventricular steatosis), myofibrillar derangement and destruction, and mitochondrial hypertrophy, as revealed by transmission electron microscopy. The detrimental effects of e-cigarettes (2.4% nicotine) on cardiac structure and function was accompanied by increased oxidative stress, plasma free fatty acid levels, CM apoptosis, and inactivation of AMP-activated protein kinase and activation of its downstream target, acetyl-CoA-carboxylase. Our results indicate profound adverse effects of e-cigarettes (2.4% nicotine) on the heart in obese mice and raise questions about the safety of the nicotine e-cigarettes use., Competing Interests: The authors have declared that no competing interests exist.

Background: E-cigarette and other novel electronic nicotine delivery systems (ENDS) have recently entered the market at a rapid pace. The community desperately needs answers about the health effects of ENDS. The present study tested the hypothesis that perinatal nicotine exposure (PNE) causes a gender-dependent increase in vulnerability of the heart to ischemia-reperfusion (I/R) injury and cardiac dysfunction in male rat offspring via reprogramming of the miRNA-181a (miR-181a)-mediated signaling pathway and that miR-181a antisense could rescue this phenotype. Methods: Nicotine or saline was administered to pregnant rats via subcutaneous osmotic minipumps from gestational day 4 until postnatal day 10. Cardiac function and molecular biological experiments were conducted in ~3- month-old offspring. Results: PNE enhanced I/R-induced cardiac dysfunction and infarction in adult male but not in female offspring, which was associated with miR-181a over-expression in left ventricle tissues. In addition, PNE enhanced offspring cardiac angiotensin receptor (ATR) expressions via specific CpG hypomethylation of AT 1 R/AT 2 R promoter. Furthermore, PNE attenuated cardiac lncRNA H19 levels, but up-regulated cardiac TGF-β/Smads family proteins and consequently up-regulated autophagy-related protein (Atg-5, beclin-1, LC3 II, p62) expression in the male offspring. Of importance, treatment with miR-181a antisense eliminated the PNE's effect on miR-181a expression/H19 levels and reversed PNE-mediated I/R-induced cardiac infarction and dysfunction in male offspring. Furthermore, miR-181a antisense also attenuated the effect of PNE on AT 1 R/AT 2 R/TGF-β/Smads/autophagy-related biomarkers in the male offspring. Conclusion: Our data suggest that PNE could induce a reprogramming of cardiac miR-181a expression/DNA methylation pattern, which epigenetically modulates ATR/TGF-β/autophagy signaling pathways, leading to gender-dependent development of ischemia-sensitive phenotype in postnatal life. Furthermore, miR-181a could severe as a potential therapeutic target for rescuing this phenotype., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country's mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABA A receptor-mediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders., (Copyright © 2020 Silva, Shao, Shen, Davies, Olsen, Holschneider, Shao and Liang.)

Objective: To observe the direct intervention effects of electroacupuncture (EA) and non-steroid anti-inflammatory drugs (NSAIDs) on pain memory, and to explore their effects on cAMP/PKA/cAMP pathway in anterior cingulate gyrus (ACC)., Methods: Fifty clean healthy male SD rats were randomly divided into a control group, a model group, an indomethacin group, an EA group and a sham EA group, 10 rats in each group. Except the control group, the pain memory model was established in the remaining four groups by twice injection of carrageenan at foot; 0.1 mL of 2%λ-carrageenan was subcutaneously injected at the left foot of rats; 14 days later, when the pain threshold of rats of each group returned to the basic level, the second injection was performed with the same procedure. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36) for 30 min; the rats in the indomethacin group was treated with indomethacin intragastric administration with the dose of 3 mg/kg; the rats in the sham EA group was treated with EA without electricity at the point 0.3 mm forward "Zusanli" (ST 36) with the depth of 2 mm for 30 min; the rats in the control group was not given any invention. All the above interventions were performed 5 h, 1 d, 2 d and 3 d after the second injection of 2% λ-carrageenan. The left-side paw withdrawal thresholds (PWT) were observed before the first injection, 4 h, 3 d, 5 d after the first injection, before the second injection and 4 h, 1 d, 2 d, 3 d after the second injection. Three days after the second injection, the number of positive cells of cAMP, p-PKA, p-CREB and the number of positive cells of protein co-expression in the right ACC brain area were detected by immunofluorescence, and the relative protein expression of p-PKA and p-CREB were detected by Western blot., Results: Compared with the control group, the PWTs in the model group decreased significantly 4 h, 3 d and 5 d after the first injection and 1 d, 2 d and 3 d after the second injection ( P <0.05); compared with the control group, the positive expression of cAMP, p-PKA and p-CREB in the right ACC brain area in the model group increased significantly ( P <0.05), and the number of positive cells of the co-expression of cAMP/p-PKA and p-PKA/p-CREB also increased significantly ( P <0.05). Compared with the model group, indomethacin group and sham EA group, the PWTs in the EA group were increased significantly 1 d, 2 d and 3 d after the second injection ( P <0.05); compared with the model group, indomethacin group and sham EA group, the positive expression of p-PKA and p-CREB in the right ACC brain area in the EA group decreased significantly ( P <0.05), and the number of positive cells of co-expression of cAMP/p-PKA and p-PKA/p-CREB was decreased significantly ( P <0.05). Compared with the model group and sham EA group, the positive expression of cAMP in the right ACC brain area was decreased in the EA group ( P <0.05)., Conclusion: EA have a direct intervention effect on pain memory, which have significant advantage over NSAIDs in the treatment of chronic pain. The advantage effect of EA on pain memory may be related to the inhibition of cAMP/PKA/CREB pathway in ACC area.

Computational prediction of binding between neoantigen peptides and major histocompatibility complex (MHC) proteins can be used to predict patient response to cancer immunotherapy. Current neoantigen predictors focus on in silico estimation of MHC binding affinity and are limited by low predictive value for actual peptide presentation, inadequate support for rare MHC alleles, and poor scalability to high-throughput data sets. To address these limitations, we developed MHCnuggets, a deep neural network method that predicts peptide-MHC binding. MHCnuggets can predict binding for common or rare alleles of MHC class I or II with a single neural network architecture. Using a long short-term memory network (LSTM), MHCnuggets accepts peptides of variable length and is faster than other methods. When compared with methods that integrate binding affinity and MHC-bound peptide (HLAp) data from mass spectrometry, MHCnuggets yields a 4-fold increase in positive predictive value on independent HLAp data. We applied MHCnuggets to 26 cancer types in The Cancer Genome Atlas, processing 26.3 million allele-peptide comparisons in under 2.3 hours, yielding 101,326 unique predicted immunogenic missense mutations (IMM). Predicted IMM hotspots occurred in 38 genes, including 24 driver genes. Predicted IMM load was significantly associated with increased immune cell infiltration ( P < 2 × 10 -16 ), including CD8 + T cells. Only 0.16% of predicted IMMs were observed in more than 2 patients, with 61.7% of these derived from driver mutations. Thus, we describe a method for neoantigen prediction and its performance characteristics and demonstrate its utility in data sets representing multiple human cancers., (©2019 American Association for Cancer Research.)

Rationale: Cigarette smoking is a well-established risk factor for myocardial infarction and sudden cardiac death. The deleterious effects are mainly due to nicotine, but the mechanisms involved and theranostics remain unclear. Thus, we tested the hypothesis that nicotine exposure increases the heart sensitivity to ischemia/reperfusion injury and dysfunction, which can be rescued by autophagy inhibitor. Methods: Nicotine or saline was administered to adult rats via subcutaneous osmotic minipumps in the absence or presence of an autophagy inhibitor, 3-methyladenine (3-MA). After 30 days of nicotine treatment, the rats underwent the cardiac ischemia/reperfusion (I/R) procedure and echocardiography analysis, and the heart tissues were isolated for molecular biological studies. Results: Nicotine exposure increased I/R-induced cardiac injury and cardiac dysfunction as compared to the control. The levels of autophagy-related proteins including LC3 II, P62, Beclin1, and Atg5 were upregulated in the reperfused hearts isolated from nicotine-treated group. In addition, nicotine enhanced cardiac and plasma ROS production, and increased the phosphorylation of GSK3β (ser9) in the left ventricle tissues. Treatment with 3-MA abolished nicotine-mediated increase in the levels of autophagy-related proteins and phosphorylation of GSK3β, but had no effect on ROS production. Of importance, 3-MA ameliorated the augmented I/R-induced cardiac injury and dysfunction in the nicotine-treated group as compared to the control. Conclusion: Our results demonstrate that nicotine exposure enhances autophagy signaling pathway, resulting in development of ischemic-sensitive phenotype of heart. It suggests a potentially novel therapeutic strategy of autophagy inhibition for the treatment of ischemic heart disease., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objective: To observe the expression of GABA A receptor mRNA in different brain regions of the central nervous system in chronic inflammatory pain rats and the intervention effect of electroacupuncture (EA)., Methods: A total of 48 SPF male SD rats were randomly divided into a blank control group, a model control group, an EA group and a sham EA group, 12 rats in each group. The model of chronic inflammatory pain was established by injecting Freund's complete adjuvant into the foot. The EA group was treated with EA 28 days after the model establishment. The "Housanli" (ST 36) and "Kunlun" (BL 60) were selected and treated with dilatational wave, 2 Hz/100 Hz in frequency, 0.5-1.5 mA for 30 min; EA was given only once. In the sham EA group, the same acupoints were selected but the needles were only inserted into subcutaneous area; EA was connected for 30 min without electrical stimulation. The behavior changes of mechanical pain threshold and thermal pain threshold before model establishment, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after the model establishment as well as emotional behavior 29 days after the model establishment were observed; the relative expressions of GABA A receptor mRNA in anterior cingulate cortex, amygdala and hypothalamus were observed., Results: Compared with the blank control group, the change rates of mechanical pain threshold and thermal pain threshold in the model control group were decreased significantly 1 day, 3 days, 7 days, 14 days, 21 days, 28 days after model establishment ( P <0.01); 29 days after model establishment, the movement distance and staying time in the central area of open field test in the model control group were decreased significantly ( P <0.05). After EA intervention, compared with the model control group and the sham EA group, the change rates of mechanical pain threshold and thermal pain threshold, as well as the movement distance and the staying time of central area were significantly increased in the EA group ( P <0.01, P <0.05). Twenty-nine days after model establishment, the expression of GABA A receptor mRNA in anterior cingulate cortex and hypothalamus was not significantly different among all groups ( P >0.05). Compared with the blank control group, the expression of GABA A receptor mRNA in the amygdala was decreased significantly in the model control group ( P <0.01); compared with the model control group and the sham EA group, the expression of GABA A receptor mRNA in amygdala was increased after intervention in the EA group ( P <0.01)., Conclusion: Single treatment of EA could significantly increase the mechanical pain threshold and thermal pain threshold, improve abnormal emotional behavior in rats with chronic inflammatory pain, which may be related to the increasing of expression of GABA A receptor mRNA in the amygdala.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport ( NH 4 + , NH 3 , or NH 3 -2H + transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H + (OH - ) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H + (OH - ) transport in the absence of ammonia. We quantitated H + flux by monitoring changes in intracellular pH (pH i ) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH 4 Cl. Our results demonstrate that SLC4A11 mediated conductive H + (OH - ) transport that was stimulated by raising the extracellular pH (pH e ). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pH e . In studies using increasing NH 4 Cl concentrations with equal NH 4 + extracellular and intracellular concentrations, the shift in the reversal potential ( E rev ) due to the addition of ammonia was compatible with NH 3 -H + transport competing with H + (OH - ) rather than NH 3 -nH + ( n ≥ 2) transport. The increase in equivalent H + (OH - ) flux observed in the presence of a transcellular H + gradient was also compatible with SLC4A11-mediated NH 3 -H + flux. The NH 3 versus E rev data fit a theoretical model suggesting that NH 3 -H + and H + (OH - ) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H + (OH - ) transport and ammonia-induced whole cell currents were blocked suggesting that the H + (OH - ) and NH 3 -H + transport processes share common features involving the SLC4A11 transport mechanism.