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Summary Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.

MicroRNA-221 (miRNA-221) is upregulated in several malignant tumors and is associated with poor patient prognosis. Therefore, the present study aimed to investigate the role and underlying mechanism of miRNA-221 in doxorubicin (DOX) resistance in osteosarcoma cells. We constructed DOX-resistant Saos-2/DOX cells and treated them with DOX. Cell viability was determined by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were transfected with either miRNA-221 mimic or miRNA-221 inhibitor; quantitative (q)RT-PCR was performed to detect the expression of miRNA-221. Flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) staining were used to detect cell apoptosis. The immunofluorescence method was also used to detect cell signal transduction and activator of transcription 3 (Stat3) protein expression distribution. In addition, Western blotting was used to detect changes in the expression of each protein. We found that miRNA-221 was upregulated in Saos-2/DOX cells. Moreover, the miRNA-221 mimic induced DOX resistance in Saos-2 cells, whereas the miRNA-221 inhibitor enhanced DOX sensitivity in Saos-2/DOX cells. The miRNA-221 mimic upregulated the expression of phosphorylated-Stat3, P-glycoprotein (P-gp), and B-cell lymphoma-2 (Bcl-2) proteins in Saos-2 cells and induced the entry of Stat3 into the nucleus, whereas the miRNA-221 inhibitor exerted the opposite effect. Pretreatment with the Stat3 chemical inhibitor, STAT3-IN-3, significantly inhibited the upregulation of P-gp and Bcl-2 protein expression induced by the miRNA-221 mimic in Saos-2 cells; it also caused the Saos-2 cells to overcome DOX resistance induced by the miRNA-221 mimic. Thus, miRNA-221 increased the expression of P-gp and Bcl-2 by activating the Stat3 pathway to promote DOX resistance in osteosarcoma cells, indicating a potential use of miRNA-221 in osteosarcoma treatment.

Background: Increased IL-6 level, M2 macrophages and PD-1+CD8+ T cells in tumor microenvironments (TME) have been identified to correlate with resistance to checkpoint blockade immunotherapy, yet the mechanism remains poorly understood. Rab small GTPase-mediated trafficking of cytokines is critical in immuno-modulation. We have previously reported dysregulation of Rab37 in lung cancer cells, whereas the roles of Rab37 in tumor-infiltrating immune cells and cancer immunotherapy are unclear. Methods: The tumor growth of the syngeneic mouse allograft in wild type or Rab37 knockout mice was analyzed. Imaging analyses and vesicle isolation were conducted to determine Rab37-mediated IL-6 secretion. STAT3 binding sites at PD-1 promoter in T cells were identified by chromatin immunoprecipitation assay. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37, IL-6 and PD-1 and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from lung cancer patients. Results: We revealed that Rab37 regulates the secretion of IL-6 in a GTPase-dependent manner in macrophages to trigger M2 polarization. Macrophage-derived IL-6 promotes STAT3-dependent PD-1 mRNA expression in CD8+ T cells. Clinically, tumors with high stromal Rab37 and IL-6 expression coincide with tumor infiltrating M2-macrophages and PD1+CD8+ T cells that predicts poor prognosis in lung cancer patients. In addition, lung cancer patients with an increase in plasma IL-6 level are found to be associated with immunotherapeutic resistance. Importantly, combined blockade of IL-6 and CTLA-4 improves survival of tumor-bearing mice by reducing infiltration of PD1+CD8+ T cells and M2 macrophages in TME. Conclusions: Rab37/IL-6 trafficking pathway links with IL-6/STAT3/PD-1 transcription regulation to foster an immunosuppressive TME and combined IL-6/CTLA-4 blockade therapy exerts potent anti-tumor efficacy.

Background: Huangqi Guizhi Wuwu decoction (HQGZWW) exhibits good effects when administered to treat multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Understanding the precise mechanism of this decoction is thus important. Based on the findings of our previous study, the aim of the present study was to understand the role of antigen-specific CD8+ T-cells on the pathogenesis of MS/EAE when HQGZWW is administered as treatment. Methods: Myelin oligodendrocyte glycoprotein (MOG) 35-55-induced mice were administered distilled water, prednisone, and high dose or low dose HQGZWW. After purified CD4+ and CD8+ T-cells were stimulated with the MOG35-55 peptide, proliferation and cytokine secretion assays were performed. To establish the adoptive transfer EAE model, naive mice were injected with MOG35-55 -CD8+ or CD4+ T-cells. Results: Significant improvements in EAE score and pathology were observed in the high dose HQGZWW and prednisone groups. Compared to the low dose HQGZWW and distilled water groups, lower antigen-specific responses, lower levels of interferon-gamma, and higher levels of interleukin (IL)-4 and IL-10 from CD8+ and CD4+ T cells were observed in the high dose HQGZWW and prednisone groups. Finally, the EAE score was observed to be similar between the high dose HQGZWW group and prednisone group; however, this finding was not observed in the low dose HQGZWW group. Conclusion: Our findings suggest that high dose HQGZWW has similar effects on cell proliferation, cytokine secretion, and EAE score to prednisone, while low dose HQGZWW does not have such effect. The protective role of HQGZWW against EAE might thus depend on the Th2 cytokine secretion profile induced by either MOG35-55 specific CD8+ or CD4+ T-cells.

Background Pasteurella multocida is an important and old zoonotic pathogen worldwide which has an impressive host spectrum including numerous domestic and wild animals as well as birds, causing specific diseases or outbreak with great economic impact. It has never been reported that P. multocida can cause an epidemic in wild rodents. In June 5–17, 2016, more than 1000 rodent deaths of an unknown cause quickly spread in the PuEr City, Yunnan province, southwestern China. Methods The rodents in affected areas and outside of the epidemic areas were collected and screened for possible known pathogens including Yersinia pestis, rabies virus and hantavirus as well as other bacteria. The possible bacterial pathogens were isolated both by culture medium and by mouse inoculation in parallel. The isolates were identified by the Vitek GNI card and PCR assays for 16S rRNA genes. The pathogen strains were selected for whole genome sequencing analysis. Results A total of 123 rodents were collected from 25 sample sites at affected area, among of which, all 119 dead rodents were negative for the pathogen under consideration except P. multocida, and all four live rodents were negative for P. multocida. In addition, 480 rodents collected from other 23 counties outside of the epidemic area in Yunnan were negative for with P. multocida. A total of 14 strains of P. multocida (six directly isolated from the field rodents and eight from the experimental mice that were injected with the organ substrates from the dead rodents) belonged to serogroup A and serogroup F represented by 9 N and 20 N were identified in these epidemic areas. Whole genome sequencing revealed that the serogroup F strain shared 99% similarity to P. multocida Pm70 from chicken, but contained a 50 k bp insertion sequence. The serogroup A strain shared 95% similarity to P. multocida FDAARGOS_385 from a human patient, but contained four large structural differences. Histological abnormalities were identified in the livers, lungs, hearts and brains of the inoculated mice. Conclusions The simultaneous occurrence of both serotypes of P. multocida may have caused this sudden onset of mortality across the local rodent population in Yunnan Province, China. Further attention should be paid to this old bacterium in the world.

PURPOSE An inherited basis for presumed sporadic neuroendocrine tumor (NET) has been suggested by evidence of familial clustering of NET and a higher incidence of second malignancies in patients and families with NET. To further investigate a potential heritable basis for sporadic neuroendocrine tumors, we performed multigene platform germline analysis to determine the frequency of hereditary susceptibility gene variants in a cohort of patients with sporadic small intestine NET (SI-NET). METHODS We performed a multigene platform germline analysis with Invitae's 83-gene, next-generation sequencing panel using DNA from 88 individuals with SI-NET from a clinically annotated database of patients with NET evaluated at Dana-Farber Cancer Institute (DFCI) who are considered high risk for inherited variants. Additionally, we evaluated the prevalence of pathogenic variants in an unselected cohort of patients with SI-NET who underwent testing with Invitae. RESULTS Of the 88 patients in the DFCI cohort, a pathogenic germline variant was identified in eight (9%) patients. In an independent cohort of 120 patients with SI-NET, a pathogenic germline variant was identified in 13 (11%) patients. Pathogenic variants were identified in more than one patient in the following genes: ATM, RAD51C, MUTYH, and BLM. Somatic testing of tumors from the DFCI cohort was suboptimal because of insufficient coverage of all targeted exons, and therefore, analysis was limited. CONCLUSION We demonstrate a 9%-11% incidence of pathogenic germline variants in genes associated with inherited susceptibility for malignancy not previously described in association with SI-NET. The association of these germline variants with neuroendocrine carcinogenesis and risk is uncertain but warrants further characterization.

Decidualization is essential for successful pregnancy in rodents and primates. Although L-Tryptophan and its metabolites are essential for mammalian pregnancy, the underlying mechanism is poorly defined. We explored effects of tryptophan and kynurenine on human in vitro decidualization in human endometrial stromal cell line and primary endometrial stromal cells. Tryptophan significantly stimulates the expression of prolactin and insulin growth factor binding protein 1, reliable markers for human decidualization. When stromal cells are treated with tryptophan, tryptophan hydroxylase-1 remains unchanged, but indoleamine 2,3-dioxygenase 1 is significantly increased, suggesting tryptophan is mainly metabolized through kynurenine pathway. Kynurenine significantly stimulates insulin growth factor binding protein 1 expression. Aryl hydrocarbon receptor and its target genes (P450 1A1 and P450 1B1) are significantly increased by tryptophan and kynurenine. The induction of tryptophan and kynurenine on insulin growth factor binding protein 1 is abrogated by CH223191, an aryl hydrocarbon receptor inhibitor. Cytochrome P450 1A1 and P450 1B1 catalyze the oxidative metabolism of estradiol to catechol estrogens (2-hydroxy estradiol and 4-hydroxy estradiol), respectively. Insulin growth factor binding protein 1 is up-regulated by 2-hydroxy estradiol and 4-hydroxy estradiol. Interferon-γ significantly induces the expression of indoleamine 2,3-dioxygenase 1, aryl hydrocarbon receptor and insulin growth factor binding protein 1. All the data are also verified in primary human stromal cells. Our data indicate that Interferon-γ-induced kynurenine pathway promotes human decidualization via aryl hydrocarbon receptor signaling.

Clinical studies have indicated that the long-term use of topical antiglaucoma drugs, such as carbonic anhydrase inhibitors (CAIs), may lead to meibomian gland dysfunction (MGD). We hypothesize that these adverse effects involve a direct influence on human MG epithelial cells (HMGECs). The purpose our present investigation was to test our hypothesis and determine whether exposure to dorzolamide, a CAI, impacts the proliferation, intracellular signaling and differentiation of HMGECs.We cultured immortalized (i) HMGECs with vehicle or various concentrations of dorzolamide for 6 days. Cells were enumerated with a hemocytometer, and examined for their morphology, Akt signaling activity, accumulation of neutral lipids, phospholipids and lysosomes, and the expression of protein biomarkers for lipogenesis regulation, lysosomes and autophagosomes.Our results show that a high, 500 µg/ml concentration of dorzolamide causes a significant decrease in Akt signaling and the proliferation of iHMGECs. However, the high dose of dorzolamide also promotes the differentiation of iHMGECs. This response features increases in the number of lysosomes, the accumulation of phospholipids, and the expression of the light chain 3A biomarker for autophagosomes. In contrast, the therapeutic amount (50 µg/ml) of dorzolamide has no impact on the proliferative or differentiative abilities of iHMGECs.Our results support our hypothesis and demonstrate that the CAI dorzolamide does exert a direct influence on the proliferation and differentiation of iHMGECs. However, this effect is elicited only by a high, and not a therapeutic, amount of dorzolamide.

Two naphthoquinone-derived heterodimers with unprecedented carbon skeletons, eleucanainones A (1) and B (2), were isolated from the bulbs of Eleutherine americana. Their structures were elucidated ...

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland’s Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts. KEY POINTS: • A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%. • The optimized three-module method improves the integration efficiency by more than three times. • This method will facilitate the functional genomic analysis and metabolic flux regulation.

As small conserved RNAs without a coding function, microRNAs are expressed in multicellular organisms and contribute to the modulation of multiple cellular reactions, such as viral replication, as well as autophagy. microRNAs can regulate host gene expression and inhibit or reinforce hepatitis B virus (HBV) replication. Hepatic cells express miR-155 noticeably. Consequently, our study explored miR-155 modulation of HBV replication and investigated the potential mechanism involved. miR-155 was inhibited on HBV infection. miR-155 transfection remarkably reinforced HBV replication, antigen expression, and progeny secretion in HepG2215 cells. Moreover, miR-155 impaired the inhibition of the cytokine signalling 1 (SOCS1)/Akt/mTOR axis and reinforced HepG2215 autophagy. Additionally, the autophagy inhibitor (3-MA) eliminated HBsAg secretion triggered by miR-155. Taken together, miR-155 reinforced HBV replication by reinforcing SOCS1-triggered autophagy. SIGNIFICANCE OF THE STUDY: The research studied the potential mechanism involved in HBV replication and miR-155 that miR-155 reinforces HBV replication by reinforcing the SOCS1/Akt/mTOR axis-stimulated autophagy, and therefore, it can provide medical practitioners with the inspiration that chronic HBV might be cured or improved by regulating the activation of miR-155 in cells. In the study, the experiments show that autophagy inhibitors (3-MA) counteracted miR-155 contribution to HBV replication, and it might be a practicable way to improve HBV through some therapies that can repress the autophagy in related cells.

Saccharum spontaneum is a wild germplasm resource of the genus Saccharum that has many valuable traits. Ty1-copia retrotransposons constitute a large proportion of plant genomes and affect genome sequence organization and evolution. This study aims to analyze the sequence heterogeneity, phylogenetic diversity, copy number, and chromosomal dispersion patterns of Ty1-copia retrotransposons in S. spontaneum. A total of 44 Ty1-copia reverse transcriptase subclones isolated from S. spontaneum showed a range of heterogeneity, and all sequences were A-T rich, averaging approximately 54.59%. Phylogenetic analysis divided the 44 reverse transcriptase sequences into 5 distinct lineages (Retrofit/Ale, Sire/Maximus, Bianca, Tork/TAR, and Ty1-copia like). Dot-blot hybridization revealed that Ty1-copia retrotransposons consisted of a significant component of approximately 38,900 copies and 16,300 copies per genome in the accessions YN82-114 (2n = 10x = 80) and AP85-441 (2n = 4x = 32), respectively. The results of a local blast analysis showed that there are 15,069 Ty1-copia retrotransposon copies in the genome of AP85-441, of which the Retrofit/Ale lineage had the highest copy number, followed by the Tork/TAR, Sire/Maximus, and Bianca lineages. Furthermore, both FISH and the local blast analysis with AP85-441 genomic data demonstrated that the Ty1-copia retrotransposons were unevenly distributed throughout the chromosomes. Taken together, this study provides insights into the role of Ty1-copia retrotransposons in the evolution and organization of the S. spontaneum genome.

Glucocorticoids (GCs) are essential for mouse embryo implantation and decidualization. Excess GCs are harmful for mouse embryo implantation and decidualization. 11β-Hydroxysteroid dehydrogenases type I and II (Hsd11b1/Hsd11b2) are main enzymes for regulating local level of GCs. Hsd11b2 acts as the placental glucocorticoid barrier to protect the fetus from excessive exposure. Although effects of GCs on the fetus and placenta in late pregnancy have been extensively studied, the effects of these adrenal corticosteroids in early pregnancy are far less well defined. Therefore, we examined the expression, regulation and function of Hsd11b1/Hsd11b2 in mouse uterus during early pregnancy. We found that Hsd11b2 is highly expressed in endometrial stromal cells on days 3 and 4 of pregnancy and mainly upregulated by progesterone (P4). In both ovariectomized mice and cultured stromal cells, P4 significantly stimulates Hsd11b2 expression. P4 stimulation of Hsd11b2 is mainly mediated by the Ihh pathway. The uterine level of corticosterone (Cort) is regulated by Hsd11b2 during preimplantation. Embryo development and the number of inner cell mass cells are suppressed by Cort treatment. These results indicate that P4 should provide a low Cort environment for the development of preimplantation mouse embryos by promoting the expression of uterine Hsd11b2.

Background: The pathogenesis of multiple sclerosis (MS) is mediated primarily by T cells, but most studies of MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have focused on CD4+ T cells. The aims of the current study were to determine the pathological interrelationship between CD4 and CD8 autoreactive T cells in MS/EAE. Methods: Female C57BL/6 mice (n =20) were induced by myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. At 14 days after immunization, T cells were isolated from the spleen and purified as CD4+ and CD8 + T cells by using CD4 and CD8 isolation kits, and then the purity was determined by flow cytometric analysis. These cells were stimulated by MOG35–55 peptide and applied to proliferation assays. The interferon-gamma (IFN-γ) and interleukin (IL)-4 secretion of supernatant of cultured CD4 + and CD8 + T cells were measured by enzyme-linked immunosorbent assays (ELISA). For adoptive transfer, recipient mice were injected with MOG35–55-specific CD8+ or CD4+ T cells. EAE clinical course was measured by EAE score at 0–5 scale and spinal cord was examined by staining with hematoxylin and eosin and Luxol fast blue staining. Results: CD8+CD3+ and CD4+CD3 + cells were 86% and 94% pure of total CD3+ cells after CD8/CD4 bead enrichment, respectively. These cells were stimulated by MOG35–55 peptide and applied to proliferation assays. Although the CD8+ T cells had a generally lower response to MOG35–55 than CD4+ T cells, the response of CD8+ T cells was not always dependent on CD4. CD8+ T cell secreted less IFN-γ and IL-4 compared with CD4+ T cells. EAE was induced in wildtype B6 naive mice by adoptive transfer of MOG35–55-specific T cells from B6 active-induced EAE (aEAE) mice. A similar EAE score and slight inflammation and demyelination were found in naive B6 mice after transferring of CD8 + T cells from immunized B6 mice compared with transfer of CD4 + T cells. Conclusion: Our data suggest that CD8+ autoreactive T cells in EAE have a lower encephalitogenic function but are unique and independent on pathogenic of EAE rather than their CD4+ counterparts. Key words: Autoimmune; CD8-Positive T-Lymphocytes; Encephalomyelitis; Experimental; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein

The wild Chinese grapevines (Vitis spp.) show varying levels of resistance to powdery mildew caused by Erysiphe necator that is an economically important disease of cultivated grapevines (Vitis vinifera). However, little information is available regarding the cytological mechanisms of powdery mildew resistance in these wild relatives. Here, we studied the cytological responses of three wild Chinese grapevine accessions after they were infected with E. necator (En) NAFU1 in comparison to the susceptible V. vinifera cv. 'Thompson Seedless' grape. The hyphal growth and sporulation of En NAFU1 were significantly restricted in wild species compared to 'Thompson Seedless', which appears to be associated with early cell wall deposition at the attempt sites, encasement of haustoria, and hypersensitive response-like cell death of penetrated epidermal cells. Moreover, endogenous free salicylic acid (SA) was more abundant in wild Chinese Vitis species than in 'Thompson Seedless' under pathogen-free condition. During En NAFU1 colonization, SA conjugates accumulated higher in wild grapevines than in 'Thompson Seedless'. In addition, the species-specific expression patterns of defense-associated genes during En NAFU1 colonization indicated that mechanisms underlying powdery mildew resistance are divergent among different wild Chinese Vitis species. These results contribute to understanding of mechanisms underlying defense responses of wild Chinese Vitis species against powdery mildew.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1β, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Vibrio alginolyticus, like other vibrio species, is a widely distributed marine bacterium that is able to outcompete other species in variable niches where diverse organic matters are supplied. However, it remains unclear how these cells sense and adjust metabolic flux in response to the changing environment. CsrA is a conserved RNA-binding protein that modulates critical cellular processes such as growth ability, central metabolism, virulence, and the stress response in gamma-proteobacteria. Here, we first characterize the csrA homolog in V. alginolyticus. The results show that CsrA activates swarming but not swimming motility, possibly by enhancing the expression of lateral flagellar associated genes. It is also revealed that CsrA modulates the carbon and nitrogen metabolism of V. alginolyticus, as evidenced by a change in the growth kinetics of various carbon and nitrogen sources when CsrA is altered. Quantitative RT-PCR shows that the transcripts of the genes encoding key enzymes involved in the TCA cycle and amino acid metabolism change significantly, which is probably due to the variation in mRNA stability given by CsrA binding. This may suggest that CsrA plays an important role in sensing and responding to environmental changes.

Background: Brahmophthalma hearseyi (Lepidoptera: Brahmaeidae) is widely distributed across China. Its larvae damage the leaves of many plants such as those belonging to the Oleaceae family, causing significant economic losses and seriously affecting the survival and reproduction of Cervus nippon, however, genetic data for this species are scarce. Methods: The complete mitochondrial genome (mitogenome) of B. hearseyi was sequenced using long-PCR and primer-walking methods. Phylogenetic analysis that was based on 13 PCGs and two rRNAs was carried out using the neighbor-joining and Bayesian interference methods. Results: The mitogenome is a typical circular molecule that is made up of 15,442 bp, which includes 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and an A + T-rich region (456 bp). All of the PCGs, except for COX1 and COX2, start with ATN codons. COX2 and ND5 use the incomplete termination codon T, and 11 other PCGs use the typical stop codon TAA. All tRNA genes, except for trnS1 and trnS2, display a typical cloverleaf structure, trnS1 lacks the “DHU” arm, whereas trnS2 exhibits two mismatched base pairs in the anticodon stem. Phylogenetic analysis showed that B. hearseyi is clustered into Brahmaeidae, and the phylogenetic relationships are (Brahmaeidae + Lasiocampidae) + (Bombycidae + (Sphingidae + Saturniidae)). Conclusions: This study provides the first mitogenomic resources for the Brahmaeidae.

BACKGROUND Plant diseases have been gripping agricultural production, seriously affecting the growth and yields of crops. Marine natural products are an important source for novel drugs discovery. In this work, pityriacitrin marine alkaloids were selected as the parent structures. A series of pityriacitrin alkaloid analogues were rationally designed, synthesized and evaluated for their antiviral activities and fungicidal activities. RESULT Most of these compounds were demonstrated to have higher antiviral activities than ribavirin. Particularly, compounds 3a, 3e, 8f, 8g, and 9g displayed higher anti-TMV activities than ningnanmycin at 500 μg·mL-1 . Mechanism research revealed that 3a could bind to TMV CP with an excellent affinity (Ka = 8.67 × 106 L·mol-1 ), thus interfere with the assembly of virus particles. These alkaloids also showed broad-spectrum fungicidal activities against eight kinds of phytopathogenic fungi. Compound 5f with 1.43-3.84 μg·mL-1 EC50 value against three fungi emerged as a new fungicidal candidate. CONCLUSION Pityriacitrin alkaloids and their derivatives were synthesized and evaluated for anti-TMV and fungicidal activities for the first time. Compounds 3a and 5f with excellent activities emerged as new candidates for antiviral research and fungicidal research, respectively. Current work provided a new idea for the molecular design and development of novel plant virus and fungi inhibitors in the future. © 2021 Society of Chemical Industry.

KDM4A is a histone lysine demethylase that has been described as an oncogene in various types of cancer. The importance of KDM4A-mediated epigenetic regulation in tumorigenesis is just emerging. Here, by using Kaposi’s sarcoma associated herpesvirus (KSHV) as a screening model, we identified 6 oncogenic virus-induced long non-coding RNAs (lncRNAs) with the potential to open chromatin. RNA immunoprecipitation revealed KSHV-induced KDM4A-associated transcript (KIKAT)/LINC01061 as a binding partner of KDM4A. Integrated ChIP-seq and RNA-seq analysis showed that the KIKAT/LINC01061 interaction may mediate relocalization of KDM4A from the transcription start site (TSS) of the AMOT promoter region and transactivation of AMOT, an angiostatin binding protein that regulates endothelial cell migration. Knockdown of AMOT diminished the migration ability of uninfected SLK and iSLK-BAC16 cells in response to KIKAT/LINC01061 overexpression. Thus, we conclude that KIKAT/LINC01061 triggered shifting of KDM4A as a potential epigenetic mechanism regulating gene transactivation. Dysregulation of KIKAT/LINC01061 expression may represent a novel pathological mechanism contributing to KDM4A oncogenicity., Author summary Epigenetic regulation of chromatin structure and gene function connects genotype to phenotype and diseases. Long non-coding RNA (lncRNA) is emerging as a novel type of epigenetic regulator exhibiting diverse biological functions. Aberrant lncRNA expression is associated with various diseases, including cancer. The widespread epigenetic changes that occur during the latent-to-lytic switch of herpes virus life cycle make it an attractive model to study epigenetic regulation. Using Kaposi’s sarcoma associated herpesvirus (KSHV) as a model, we screened the epigenetic function of lncRNAs whose expression was induced by reactivation of this oncogenic virus. KIKAT/LINC01061 was identified as a novel histone lysine-specific demethylase 4A (KDM4A) interacting lncRNA. KDM4A is the first identified histone trimethyl demethylase that has been demonstrated as an oncogene in various cancers. Our data reveal a novel lncRNA-mediated regulation of the epigenetic function of KDM4A and demonstrate this lncRNA-chromatin modifier interaction may serve as a potential target in cancer therapy.

Ebola virus (EBOV), a member of the filovirus family, is an enveloped negative-sense RNA virus that causes lethal infections in humans and primates. Thousands of people have died from the Ebola virus disease (EVD) in West Africa, and no specific antiviral medication and treatment have been approved for EVD. Although the development of an EBOV vaccine is promising, immunity to any vaccine is not immediate. Here, we computationally analysed the structure of EBOV glycoprotein GP2 (GP2EBOV) and designed RNA aptamers that recognize and inhibit it. The aptamers specifically bind to conserved arginine residues (Arg587 and Arg596) located in the C-terminal coiled-coil region of GP2EBOV. Molecular docking of the synthetic RNA aptamers with the ectodomain of GP2EBOV revealed that the optimized orthogonal RNA aptamers have strong binding affinities with the coiled-coil region of GP2EBOV. The characterized RNA aptamers may facilitate strategies to block replication of EBOV and related Filoviruses, and thus may serve as important antivirals to reduce mortality associated with these infections.

Ten new cucurbitane triterpenoids, hemsleyacins A–J (1–10), together with three known cucurbitane triterpenoids, dihydrocucurbitacin F (11), scandenogenin D (12), and jinfushanencin F (13), were separated from ethanolic tuber extracts of Hemsleya penxianensis. The absolute configurations of the new compounds were established based on NMR, HRESIMS, and CD spectra. Compounds 7 and 10–12 were evaluated in terms of their antifeedant activity against Plutella xylostella larvae. The result showed that compound 10 exhibited potent antifeedant activity against P. xylostella larvae after 48 h of treatment. Furthermore, the MTT test showed that compound 11 exhibited potent inhibition toward the UMUC-3 and T24 cell lines with IC50 values of 29.12 and 35.62 μM, respectively, compared to the positive control cisplatin IC50 values of 8.27 and 13.72 μM. Western blot analysis revealed that compound 11 treatments substantially inhibited the phosphorylation of IκBα.

Temperate mixed forests, along with other high latitudinal ecosystems, are more vulnerable to global warming in comparison with warm sites, because of the slower carbon (C) turnover and higher soil organic carbon (SOC) accumulation. Lignin and cellulose are two major components of plant litter, and usually make contributions to the recalcitrant and labile SOC pool, respectively. Because the chemical composition of SOC plays key role in regulating the bioavailability of soil C pool, understanding the relationship between soil lignin or cellulose content and temperature are of great significance in evaluating the feedbacks between SOC pool and the future scenarios of global warming. The biological degradation of soil lignin or cellulose is mainly dependent on soil enzymatic activities, and thus, the response of ligninolytic and cellulolytic enzymes to increased temperature would determine C release under future warming scenarios. However, the responses of the soil lignin/cellulose content and the activity of cellulolytic and ligninolytic enzymes to increased mean annual temperature (MAT) have rarely been studied, and the factors driving these changes are not fully understood. Latitudinal gradients are often used for monitoring global-warming-related problems, because of its natural gradients of temperature. In this study, we demonstrate the latitudinal pattern of lignin/cellulose content and the activities of cellulose- and lignin- degrading enzymes in a temperate Broad-leaved Korean pine mixed forests distributed along a latitudinal gradient (with MAT ranging from −1.9 to 5.1 °C) in northeastern China. The linear mixed model revealed that soil lignin content was negatively correlated with MAT (Slope = −7.604, t = −2.608, P = 0.011), whereas soil cellulose content showed no response to increased MAT. The activity of soil polyphenol oxidase (PPO), one of the enzymes catalyze lignin decomposition, was higher in high-latitude sites, in contrast, the activity of the cellulase (CEL) complex was higher in low-latitude plots. Structural equation model (SEM) analysis indicates that MAT can directly influence soil lignin or cellulose content, and indirectly through changing NRCB, plant litter C/N, microbial biomass, and degrading enzymatic activities. The value of soil lignin/(lignin + cellulose) ratio and soil lignocellulose index (LCI, lignin/(lignin + holocellulose) ratio), varied between 0.8–0.9, and 0.6–0.8, respectively, indicating that the SOC pool in this temperate ecosystem is dominated by recalcitrant components. The negative correlations between MAT and LCI, soil lignin/(lignin + cellulose), and log (PPO + PER)/log(CEL) (Slope = −0.008, t = −2.363, P = 0.021; Slope = −0.004, t = −3.134, P = 0.003, and Slope = −0.057, t = −4.477, P

The performance and microbial community succession of a 36 L working volume plug flow reactor was evaluated for treated cattle manure at an organic loading rate of 2.67 g-TS/L/day, a temperature of 38 °C ± 1 °C, and a hydraulic retention time of 18 days. A reflux of acidizing fluid effectively enhanced anaerobic digestion performance and promoted optimization of microbial community structure. The average biogas volume production rate was 1.08 L biogas/L reactor, which was 116.5% higher than the control without reflux of acidizing fluid. The specific qmethane production yield and methane content reached 0.204 L/g VS and 70%. Moreover, methane yield achieved 0.34 m3/kg removal COD with a COD removal of about 70.56%. The bacteria genera Christensenellaceae, Bacteroidales, vadinBC27, Ruminococcaceae and Treponema_2 were further enriched. Methanosarcina became the dominant methanogen in the whole PFR operation process. This study offers new opportunities for producing renewable energy from enhanced cattle manure biodegradability.

Chronic constipation is a common gastrointestinal dysfunction, but its aetiology and pathogenesis are still unclear. Interestingly, the compositions of the gut microbiota in constipation patients and healthy controls are different. Various studies reported the different gut microbiota alterations in constipation patients, but most studies indicated that constipation patients showed the decreased beneficial bacteria and the reduced species richness of gut bacteria. Besides, the alterations in the gut microbiota may lead to constipation and constipation-related symptoms and the regulation of gut microbiota has a positive effect on gut functional diseases such as constipation. Microbial treatment methods, such as probiotics, prebiotics, synbiotics, and fecal microbiota transplantation, can be used to regulate gut microbiota. Increasing evidences have suggested that Chinese medicine (CM) has a good therapeutic effect on chronic constipation. Chinese medicine is well known for its multitarget and multimode effects on diseases as well as less side effects. In previous studies, after the treatment of constipation with CM, the gut microbiota was restored, indicating that the gut microbiota might be the target or important way for CM to exert its efficacy. In this review, we summarized the effects of microbial treatment and CM on the gut microbiota of constipation patients and discussed the relationship between CM and gut microbiota.

The 35S ribosomal DNA (rDNA) units that occur in tandem repeat are separated by an intergenic spacer (IGS) that plays an important role in rRNA transcription. Moreover, IGS is an important molecular marker for evolutionary research in plants. In the present study, the IGS sequence of Erianthus arundinaceus was isolated and sequenced for the first time. Structure analysis indicated the entire IGS sequence of three typical E. arundinaceus genotypes was highly conserved, with approximately 3087 bp and 67.1% mean GC content. The putative transcription termination, and initiation sites as well as a large number of methylation sites were found to be present in the IGS of E. arundinaceus compared to other plants. The phylogenic tree constructed using the E. arundinaceus IGS sequence showed that Miscanthus sinensis var. glaber was genetically close to Saccharum spp. while E. arundinaceus was close to Imperata cylindrica. Moreover, fluorescent in situ hybridization revealed that IGS and pTa71 probes had the same locus at nucleolar organizer regions. Taken together, this work enhances our current understanding of the organization of IGS in E. arundinaceus and provides a molecular evidence for an evolutionary relationship between Saccharum spp., E. arundinaceus, I. cylindrica and M. sinensis var. glaber.

Glucose and xylose co-fermentation is crucial to maximize hydrogen yield from waste lignocellulose. In this study, cell growth, sugar consumption, and hydrogen production profiles of Thermoanaerobacter thermosaccharolyticum W16 feeding with a range of glucose and xylose were experimental investigated coupled with kinetic analysis. Results showed although T. thermosaccharolyticum W16 could use both glucose and xylose for hydrogen production, a maximum cell growth rate of 0.27 g/L/h and hydrogen production rate of 14.53 mmol/L/h was found with glucose as sole substrate, the value was 92.8% and 49.8% higher than using xylose as the only carbon source. Further interpolation analysis and experimental demonstration suggested when glucose content in the mixed substrate higher than 58.2%, the inhibitory effect on xylose utilization was increased, but when glucose concentration fell below 21.7%, its utilization will be subject to a certain degree of feedback inhibition. Coupling experimental results with kinetic analysis in this study provides a powerful evidence to further develop the potential of T. thermosaccharolyticum W16 as a biocatalyst for hydrogen production from lignocellulosic biomass.

Lung adenocarcinoma (LAD) is the leading cause of cancer death worldwide. Long noncoding RNAs (lncRNAs) have been shown to play an important regulatory role in cancer biology, including that of LAD. The aim of this experiment was to explore the interaction of LINC00483, microRNA-144 (miR-144), and homeobox A10 (HOXA10), and their effects on radio sensitivity and epithelial-mesenchymal transition (EMT) of LAD. Initially, microarray analysis was used to screen out miRNAs and lncRNAs, as well as the differentially expressed genes related to LAD. Following the screening process, the targeting relationship of LINC00483, miR-144, and that of miR-144 and HOXA10 was determined. Following that, the expression of LINC00483, miR-144, messenger RNA (mRNA), as well as protein expression of HOXA10, MMP-2, MMP-9, E-cadherin, vimentin, and N-cadherin that followed in cells was determined. Also, the effect of LINC00483 on cell migration and invasion ability, and cell tumorigenic ability was detected. LINC00483 and HOXA10 were found to be upregulated whereas miR-144 was downregulated in LAD. Silencing of LINC00483 could competitively bind to miR-144, thereby upregulating HOXA10. LINC00483 or HOXA10 silencing led to decreased HOXA10, MMP-2, MMP-9, vimentin, and N-cadherin but elevated miR-144 and E-cadherin. Moreover, after being transfected with silenced LINC00483, the cell proliferation, migration, and invasion were inhibited with enhanced radiosensitivity. Consequently, the data of the study indicates that interference of LINC00483 weakens its competitive binding ability to miR-144, thus reducing HOXA10 expression, and enhancing radiosensitivity in LAD.

Intergeneric hybrids between Saccharum spp. and Erianthus arundinaceus and clones derived from these hybrids and backcrosses to Saccharum spp. were used to study the transmission of E. arundinaceus chromosomes by genomic in situ hybridization (GISH). True hybrid progenies were precisely identified using PCR with a primer pair, AGRP52/53. The results showed that AGRP52/53 was an E. arundinaceus-specific primer pair and could be used as molecular marker to assist breeding. EaHN92, a 364 bp E. arundinaceus-specific tandem repeat satellite DNA sequence, was cloned from the E. arundinaceus clone HN92–105 with AGRP52/53, and was localized on sub-telomeric regions of all E. arundinaceus chromosomes. YCE06–61, a BC3 progeny, had 7 E. arundinaceus chromosomes and its progenies had approximately 1–6 E. arundinaceus chromosomes. The number of E. arundinaceus chromosomes in true hybrids appeared as Gaussian distribution in 3 cross combinations. In addition, GISH detected intergeneric chromosome translocation in a few progenies. Hence, screening clones containing approximately 1–2 E. arundinaceus chromosomes without translocation could be used for sorting and sequencing E. arundinaceus chromosomes. This study provides a method for breeders to select true hybrid progenies between Saccharum spp. and E. arundinaceus, which will accelerate this intergeneric hybridization breeding.

Three novel meroterpenoids, clavipines A–C (1–3), possessing a benzoquinone fused to an azepine ring and a ten-membered carbocycle with α,β-epoxy/unsaturated-γ-lactone, along with a new natural compound clavilactone F (4) and two known clavilactones D (5) and A (6) were isolated from the basidiomycete Clitocybe clavipes. Their structures with absolute configurations were unambiguously established by extensive spectroscopic analyses, and ECD and X-ray methods. An unusual seven-azepine skeleton was postulated to be formed biosynthetically via a rearrangement of quinone–amino acid conjugates. The biosynthetic origin of meroterpenoids with a benzo-fused 10-membered carbocycle unit was determined by incorporation of 13C-labeled precursors. Compound 1 exhibited significant antiproliferative activity against HepG2 and A549 cells with IC50 values of 4.28 ± 0.26 and 7.49 ± 0.41 μM, respectively.

Drug-resistant Gram-negative bacteria are a growing threat to children; thus new antibiotics are needed to treat infections caused by these pathogens. Ceftolozane/tazobactam is active against many Gram-negative pathogens and is approved for treatment of complicated intra-abdominal and urinary tract infections in adults, but has not been evaluated in children.This phase 1, noncomparative, open-label, multicenter study characterized the pharmacokinetics (by noncompartmental analysis), safety, and tolerability of single intravenous doses of ceftolozane/tazobactam in pediatric patients (birth [7 days postnatal] to18 years of age) with proven/suspected Gram-negative infection or receiving perioperative prophylaxis (clinicaltrials.gov NCT02266706). Patients were enrolled into 1 of 6 age groups to receive a single, age-based ceftolozane/tazobactam dose, with timed blood sample collection for determining plasma concentrations of ceftolozane and tazobactam. Safety and tolerability were also evaluated.Thirty-seven patients received study drug; 34 were included in the pharmacokinetic population. Ceftolozane and tazobactam pharmacokinetic parameters were generally comparable for patients 3 months to18 years of age. Patients from birth (7 days postnatal) to3 months of age had lower clearance than older children, likely due to the immature renal function of these young infants. No deaths, study drug-related serious adverse events, or clinically significant laboratory abnormalities were observed after administration of ceftolozane/tazobactam.The doses evaluated in this study yielded ceftolozane/tazobactam exposure levels generally comparable with those in adults. Single doses of ceftolozane/tazobactam were well-tolerated, and no safety concerns were identified. These data informed pharmacokinetic/pharmacodynamic models to derive pediatric dose recommendations for phase 2 ceftolozane/tazobactam clinical trials.

Accumulating evidence indicates that post-injury inflammation characterized by activated microglia contributes much to the neuropathology of ischemic injury. Several studies have demonstrated that microglia exhibit two entirely different functional activation states, referred to as classically activated (M1) and alternatively activated (M2) phenotype. Promoting microglial phenotype to switch from M1 dominant to M2 dominant might be a promising approach for handling ischemic injury. However, the comprehensive mechanism that underlines microglia polarization in ischemic brain remains unclear. Neuronal erythropoietin-producing human hepatocellular carcinoma cell receptor 4 (EphA4), the richest Eph receptor in the central nervous system (CNS), upregulate after ischemia and may have the potential to regulate microglia activation. We hypothesized that modulating EphA4/ephrin signaling could affect ischemic injury through controlling microglia polarization. We therefore knocked down neuronal EphA4 with short hairpin RNA (shRNA) and determined the role of EphA4/ephrin signaling in oxygen-glucose deprivation and reperfusion (OGD/R)-induced injury. We found that EphA4 shRNA treatment attenuated OGD/R-induced apoptosis and microglia proliferation. Neuronal EphA4 knockdown also promoted microglial M2 polarization, which reduced pro-inflammatory mediators and released anti-inflammatory cytokines as well as neurotrophic factors. We further revealed that EphA4 shRNA treatment functioned through RhoA/Rho-associated kinase 2 (ROCK2) signaling, a key mediator of microglia alternative activation. Together, these data suggested that blockage of EphA4/ephrin signaling between neuron and microglia decreased OGD/R-induced injury by promoting alternative activation of microglia via RhoA/ROCK2 signaling.

Prostate cancer is one of the most common seen malignancies and the leading cause of cancer-related death among men. Given the importance of early diagnosis and treatment, it is worth to identify a potential novel therapeutic target for prostate cancer. Mucosa-associated lymphoid tissue 1 (MALT1) is a novel gene involved in nuclear factor κB (NF-κB) signal transduction by acting as an adaptor protein and paracaspase, with an essential role in inflammation and tumorigenesis in many cancers. This study investigated the functions and the potential regulatory mechanisms of MALT1 in the human prostate cancer cells. We found that MALT1 is abundant in prostate cancer tissues. MALT1 facilitated NF-κB subunits (p50 and p65) nuclear translocation to induce gene expression of interleukin 6 (IL-6) and C-X-C motif chemokine 5 (CXCL5) in prostate carcinoma cells. MALT1 promoted cell proliferation, invasion, and tumor growth in vitro and in vivo. MALT1 enhanced NF-κB activity in prostate carcinoma cells, moreover, NF-κB induced MALT1 expression determined by reporter and immunoblot assays, implying there is a positive feedback loop between MALT1 and NF-κB. In conclusion, MALT1 is a NF-κB-induced oncogene in the human prostate carcinoma cells.

Based on the structure of the natural product cysteine, a series of thiazolidine-4-carboxylic acids were designed and synthesized. All target compounds bearing thiazolidine-4-carboxylic acid were characterized by 1H-NMR, 13C-NMR, and HRMS techniques. The antiviral and antifungal activities of cysteine and its derivatives were evaluated in vitro and in vivo. The results of anti-TMV activity revealed that all compounds exhibited moderate to excellent activities against tobacco mosaic virus (TMV) at the concentration of 500 &mu, g/mL. The compounds cysteine (1), 3&ndash, 4, 7, 10, 13, 20,23, and 24 displayed higher anti-TMV activities than the commercial plant virucide ribavirin (inhibitory rate: 40, 40, and 38% at 500 &mu, g/mL for inactivation, curative, and protection activity in vivo, respectively), especially compound 3 (inhibitory rate: 51%, 47%, and 49% at 500 &mu, g/mL for inactivation, curative, and protection activity in vivo, respectively) with excellent antiviral activity emerged as a new antiviral candidate. Antiviral mechanism research by TEM exhibited that compound 3 could inhibit virus assembly by aggregated the 20S protein disk. Molecular docking results revealed that compound 3 with higher antiviral activities than that of compound 24 did show stronger interaction with TMV CP. Further fungicidal activity tests against 14 kinds of phytopathogenic fungi revealed that these cysteine derivatives displayed broad-spectrum fungicidal activities. Compound 16 exhibited higher antifungal activities against Cercospora arachidicola Hori and Alternaria solani than commercial fungicides carbendazim and chlorothalonil, which emerged as a new candidate for fungicidal research.

Bacillus anthracis is an extremely dangerous bacterium that is associated with high morbidity and mortality. 2,6-Pyridine dicarboxylic acid (DPA) is a major biomarker of Bacillus anthracis, and it is of great significance to be able to detect DPA in a rapid, efficient, and sensitive way. Herein, a 3D network metal–organic framework (Tb-MOF) with excellent thermal and water stability was synthesized. Tb-MOF could be used to selectively detect DPA via green fluorescence recovery with a fluorescence intensity enhancement factor of 103. In addition, due to the high detection sensitivity (a detection limit of 2.4 μM) and excellent anti-interference abilities, Tb-MOF was less affected by environmental factors when compared with a “turn-off”-response luminescence sensor; it can be employed as a promising “turn-on” luminescence sensor for DPA in the future. Finally, quantum calculations showed that a large energy difference appeared between the 5D4 level of Tb3+ and the first excited triplet energy level of H2-DHBDC2−, which was the reason that the complex did not show characteristic Tb3+ emission.

Microbial carriers may co-transport polycyclic aromatic hydrocarbons (PAHs), but lack substantial experimental evidence. Cable bacteria use gliding or twitching motility to access sulfide; hence, they could be important microbial carriers in co-transporting PAHs from the sediment-water interface into suboxic zones. In this study, the effect of cable bacteria on pyrene migration was investigated by connecting or blocking the paths of cable bacteria to the suboxic zones. The results showed that downward migration of pyrene in the connecting groups were significantly higher (17.3-49.2%, p 0.01) than those in the control groups. Meanwhile, significant downward migration of microbial communities in the connecting groups were also observed, including abundant filamentous-motile microorganisms, especially cable bacteria. The adsorption of surrounding particles by cable bacteria were morphologically evidenced. The biomechanical model based on the Peclet number indicated that filamentous-motile microorganisms demonstrated stronger adsorption ability for pyrene than other microorganisms. Supposedly, the downward migration of microbial communities, especially cable bacteria, significantly enhanced pyrene migration, thus influencing the distribution and ecological risk of pyrene in sediments. This study provides new insights into the important roles of motile microorganisms in the migration of PAHs in sediments, shedding lights on guidance for ecological risk assessment of PAHs.

In this study, an innovative approach was proposed based on the implement of biochar derived from residue cornstalk left after anaerobic bio-hydrogen production (RCA-biochar) to improve direct bio-hydrogen production from anaerobic fermentation of cornstalk. The bio-hydrogen production potential and maximum bio-hydrogen production rate increased from 156.2 to 286.1 mL H2/g substrate and 3.5 to 5.7 mL H2/g substrate/h, respectively, following the added RCA-biochar increased from 2.5 to 15.0 g/L. Cornstalk chemical component analysis showed the cellulose and hemicellulose content decreased by 17.9–33.7% and 14.4–25.2%, and lignin content increased by 20.3–42.8%, respectively, after 96 h anaerobic fermentation with RCA-biochar 2.5–15.0 g/L. Further analyses revealed that RCA-biochar not only provided more specific surface area for hydrogen-producing bacteria attachment, but also promoted the cellulolytic enzyme activity, thereby resulted in increased substrate conversion to bio-hydrogen. The findings obtained in this study may provide supports for effective and sustainable lignocellulosic bio-hydrogen production in the future.

Embryo implantation involves a sterile inflammatory reaction that is required for the invasion of the blastocyst into the decidua. Adenosine triphosphate (ATP) released from stressed or injured cells acts as an important signaling molecule to regulate many key physiological events, including sterile inflammation. We found that the amount of ATP in the uterine luminal fluid of mice increased during the peri-implantation period, and this depended on the presence of an embryo. We further showed that the release of ATP from receptive epithelial cells was likely stimulated by lactate released from the blastocyst through connexin hemichannels. The ATP receptor P2y2 was present on uterine epithelial cells during the preimplantation period and increased in the stromal cells during the time at which decidualization began. Pharmacological inhibition of P2y2 compromised decidualization and implantation. ATP-P2y2 signaling stimulated the phosphorylation of Stat3 in uterine luminal epithelial cells and the expression of early growth response 1 (Egr1) and prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox-2), all of which are required for decidualization and/or implantation, in stromal cells. Short exposure to high concentrations of ATP promoted decidualization of primary stromal cells, but longer exposures or lower ATP concentrations did not. The expression of genes encoding ATP-degrading ectonucleotidases increased in the decidua during the peri-implantation period, suggesting that they may limit the duration of the ATP signal. Together, our results indicate that the blastocyst-induced release of ATP from uterine epithelial cells during the peri-implantation period may be important for the initiation of stromal cell decidualization.

Litsea cubeba essential oil (LCEO), as naturally plant-derived products, possess good antimicrobial activities against many pathogens, but their high volatility and poor water solubility limit greatly the application in food industry. In this research, inclusion complex based on &beta, cyclodextrin (&beta, CD) and LCEO, was prepared by saturated aqueous solution method. An optimum condition using the response surface methodology (RSM) based on Box&ndash, Behnken design (BBD) was obtained with the inclusion time of 2 h and &beta, CD/LCEO ratio of 4.2 at 44 &deg, C. Under the condition, the greatest yield of 71.71% with entrapment efficiency of 33.60% and loading capacity of 9.07% was achieved. In addition, the structure and characteristic of LCEO/&beta, CD inclusion complex (LCEO/&beta, CD-IC) were investigated using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR), which indicated that LCEO/&beta, CD-IC was successfully formed. The particle size of LCEO/&beta, CD-IC was determined to be 17.852 &mu, m. Thermal properties of LCEO/&beta, CD-IC evaluated by thermogravimetric-differential scanning calorimetry (TG-DTA) illustrated better thermal stability of the aimed product compared with the physical mixture. Furthermore, the tests of antifungal activity showed that LCEO/&beta, CD-IC was able to control the growth of Penicillium italicum, Penicillium digitatum, and Geotrichum citri-aurantii isolated from postharvest citrus. Our present study confirmed that LCEO/&beta, CD-IC might be further applied as an alternative to chemical fungicides for protecting citrus fruit from postharvest disease.

Local renin-angiotensin system (RAS) in female reproductive system is involved in many physiological and pathological processes, such as follicular development, ovarian angiogenesis, ovarian, and endometrial cancer progress. However, studies on the functional relevance of RAS in human endometrium are limited, especially for renin-angiotensin-aldosterone system (RAAS). In this study, we defined the location of RAS components in human endometrium. We found that angiotensin II type-1 receptor (AT1R) and aldosterone synthase (CYP11B2), major components of RAAS, are specifically expressed in endometrial gland during mid-secretory phase. Aldosterone receptor, mineralocorticoid receptor (MR), is elevated in stroma in mid-secretory endometrium. In vitro, MR is also activated by aldosterone during decidualization. Activated MR initiates LKB1 expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. The impact of PDK4 on decidualization is independent on PDHE1α inactivation. Based on co-immunoprecipitation, PDK4 interacts with p-CREB to prevent its ubiquitination for facilitating decidualization via FOXO1. Restrain of MR activation interrupts LKB1/p-AMPK/PDK4/p-CREB/FOXO1 pathway induced by aldosterone, indicating that aldosterone action on decidualization is mainly dependent on MR stimulation. Aldosterone biosynthesized in endometrial gland during mid-secretory phase promotes decidualization via activating MR/LKB1/p-AMPK/PDK4/p-CREB/FOXO1 signaling pathway. This study provides the valuable information for understanding the underlying mechanism during decidualization.

AIM Follistatin-like-1 (FSTL-1) is considered to be a novel cytokine, and it is associated with metabolic diseases. However, it is necessary to investigate further the association of FSTL-1 with metabolic syndrome (MetS) and insulin resistance (IR). We performed a cross-sectional study to investigate the associated of circulating FSTL-1 with the MetS. MATERIALS AND METHODS A cross-sectional study was performed in 487 Chinese people, including 231 control subjects and 256 patients with MetS. Bioinformatics analysis was used to determine the protein and pathways associated with FSTL-1. The protein and protein interaction (PPI) network was constructed and analysed. Serum FSTL-1 concentrations were determined by an ELISA assay. The association of FSTL-1 with MetS components and IR was assessed. RESULTS Serum FSTL-1 levels were markedly higher in patients with newly diagnosed MetS than in controls (7.5 [5.6-9.2] vs 5.8 [5.0-7.7] μg/L, P

Background Genetic locus were identified associated with ARDS outcome. Our goal was to explore the associations between genetic variants and outcome of ARDS, and the prognostic values of nucleotide polymorphism in ARDS.Methods This was a single-center, prospective trial enrolling adult ARDS patients. After baseline data were collected, blood samples were drawn to perform whole exome sequencing, and single nucleotide polymorphism (SNP) / insertion-deletion to explore the quantitative and functional associations between genetic variants and ICU outcome. Then the lung injury burden (LIB), which was defined as the ratio of nonsynonymous SNP number per megabase of DNA, was used to evaluate its value in predicting outcome of ARDS. Results A total of 105 ARDS patients were enrolled in the study, including 70 survivors and 35 nonsurvivors. Based on the analysis of a total of 65542 nonsynonymous SNP, LIB in survivors was significantly higher than nonsurvivors [1892 (1848 - 1942) /MB versus 1864 (1829 - 1910) /MB, p = 0.018], while GO analysis showed that 60 functions were correlated with ARDS outcome, KEGG enrichment analysis showed that SNP/InDels were enriched in 13 pathways. Several new SNPs were found potentially associated with ARDS outcome. Analysis of LIB was used to determine its outcome predicting ability, the area under the ROC curve of which was only 0.6103, and increase to 0.712 when combined with APACHE II score. Conclusions Genetic variants are associated with ARDS outcome; however, their prognostic value still need to be verified by larger trials.Trial registration Clinicaltrials.gov NCT02644798. Registered 20 April 2015.

Nitrogenous sesquiterpenoids fromnatural sourcesare rare, so unsurprisingly neither the potentially valuable bioactivity nor thebroad structural diversity of nitrogenous sesquiterpenoids has been reviewed before. This report covers the progressduring the decade from 2010 to February 2020 on the isolation, identification, and bioactivity of 391 nitrogen-containing natural sesquiterpenes from terrestrial plant, marine organisms, and microorganisms. This complete and in-depth reviewshouldbe helpful for discovering and developing new drugs of medicinal valuerelated to natural nitrogenous sesquiterpenoids.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that infects humans and exhibits a biphasic life cycle consisting of latent and lytic phases. Following entry into host cells, the KSHV genome undergoes circularization and chromatinization into an extrachromosomal episome ultimately leading to the establishment of latency. The KSHV episome is organized into distinct chromatin domains marked by variations in repressive or activating epigenetic modifications, including DNA methylation, histone methylation, and histone acetylation. Thus, the development of KSHV latency is believed to be governed by epigenetic regulation. In the past decade, interrogation of the KSHV epitome by genome-wide approaches has revealed a complex epigenetic mark landscape across KSHV genome and has uncovered the important regulatory roles of epigenetic modifications in governing the development of KSHV latency. Here, we highlight many of the findings regarding the role of DNA methylation, histone modification, post-translational modification (PTM) of chromatin remodeling proteins, the contribution of long non-coding RNAs (lncRNAs) in regulating KSHV latency development, and the role of higher-order episomal chromatin architecture in the maintenance of latency and the latent-to-lytic switch.

Photothermal therapy (PTT) has shown promising potential and bright prospects in damaging primary tumors; however, it is limited to metastatic and recrudescent tumors as PTT requires straightforward light irradiation. Moreover, metastatic and recrudescent tumor immunosuppression due to host T-cell antitumor activity is dramatically impeded because of programmed cell death 1 ligand (PD-L1) and programmed cell death receptor 1 (PD-1) pathways and immune checkpoint blockade (ICB) therapy. In this work, we demonstrate that PTT combined with ICB could not only eliminate primary tumors, but also prevent tumor metastasis to the lungs/liver. In particular, we have designed immunoadjuvant nanomedicine carriers on the basis of polydopamine (PDA) simultaneously loaded with resiquimod (R848)—a kind of toll-like receptor 7 (TLR7) agonist—and carbon dots (CDs)—a fluorescent agent. This nanomedicine is defined as PDA-PEG-R848-CD nanoparticle (NP). The multitasking PDA-PEG-R848-CD NPs can destroy 4T1 breast tumors by PTT under near-infrared laser irradiation in addition to generating tumor-associated antigens. Moreover, the PTT effect triggered the release of R848, thereby inducing a strong antitumor immune response. Meanwhile, this synergistic therapy also shows the abscopal effects by completely inhibiting the growth of untreated distant tumors by effectively triggering the tumors infiltrated by CD3/CD8. Such findings suggest that PDA-PEG-R848-CD NPs could significantly potentiate the systemic therapeutic efficiency of PD-L1 checkpoint blockade therapy by activating both innate and adaptive immune systems in the body.

The association between lung and intestine has already been reported, but the differences in community structures or functions between lung and intestine bacteria yet need to explore. To explore the differences in community structures or functions, the lung tissues and fecal contents in rats were collected and analyzed through 16S rRNA sequencing. It was found that intestine bacteria was more abundant and diverse than lung bacteria. In intestine bacteria, Firmicutes and Bacteroides were identified as major phyla while Lactobacillus was among the most abundant genus. However, in lung the major identified phylum was Proteobacteria and genus Pseudomonas was most prominent genus. On the other hand, in contrast the lung bacteria was more concentrated in cytoskeleton and function in energy production and conversion. While, intestine bacteria were enriched in RNA processing, modification chromatin structure, dynamics and amino acid metabolism. The study provides the basis for understanding the relationships between lung and intestine bacteria.

Background miRNAs regulate a multitude of cellular processes and their aberrant regulation is linked to human cancer. However, the role of miR-425-5p in lung cancer (LCa) is still largely unclear. Here, we explored the role of miR-425-5p during LCa tumorigenesis. Methods Cell proliferation was evaluated by cell counting Kit-8 and colony formation assay. Western blot and real-time PCR were accordingly used to detect the relevant proteins, miRNA and gene expression. Luciferase reporter assays were used to illustrate the interaction between miR-425-5p and PTEN. Results We demonstrate that miR-425-5p is overexpressed in LCa tissue and enhances the proliferative and colony formation capacity of the LCa cell lines A549 and NCI-H1299. Through predictive binding assays, PTEN was identified as a direct gene target and its exogenous expression inhibited the pro-cancer effects of miR-425-5p. Through its ability to down-regulate PTEN, miR-425-5p activated the PI3K/AKT axis. Conclusion We conclude that miR-425-5p promotes LCa tumorigenesis through PTEN/PI3K/AKT signaling.

In mammals, microRNAs can be actively secreted from cells to blood. miR‐29b‐3p has been shown to play a pivotal role in muscle atrophy, but its role in intercellular communication is largely unknown. Here, we showed that miR‐29b‐3p was upregulated in normal and premature aging mouse muscle and plasma. miR‐29b‐3p was also upregulated in the blood of aging individuals, and circulating levels of miR‐29b‐3p were negatively correlated with relative appendicular skeletal muscle. Consistently, miR‐29b‐3p was observed in exosomes isolated from long‐term differentiated atrophic C2C12 cells. When C2C12‐derived miR‐29b‐3p‐containing exosomes were uptaken by neuronal SH‐SY5Y cells, increased miR‐29b‐3p levels in recipient cells were observed. Moreover, miR‐29b‐3p overexpression led to downregulation of neuronal‐related genes and inhibition of neuronal differentiation. Interestingly, we identified HIF1α‐AS2 as a novel c‐FOS targeting lncRNA that is induced by miR‐29b‐3p through down‐modulation of c‐FOS and is required for miR‐29b‐3p‐mediated neuronal differentiation inhibition. Our results suggest that atrophy‐associated circulating miR‐29b‐3p may mediate distal communication between muscle cells and neurons., miR‐29b‐3p‐containing exosomes released from atrophied muscle can be transported via the circulation and transferred to neuronal cells. Increased miR‐29b‐3p levels in neuronal cells may lead to inhibition of neuronal differentiation.